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Background: There are limited preclinical orthotopic prostate cancer models due to the technical complexity of surgical engraftment and tracking the tumor growth in the mouse prostate gland. Orthotopic xenografts recapitulate the tumor microenvironment, tumor stromal interactions, and clinical behavior to a greater extent than xenografts grown at subcutaneous or intramuscular sites., Methods: This study describes a novel micro-surgical technique for orthotopically implanting intact tumors pieces from cell line derived (transgenic adenocarcinoma mouse prostate [TRAMP]-C2) or patient derived (neuroendocrine prostate cancer [NEPC]) tumors in the mouse prostate gland and monitoring tumor growth using magnetic resonance (MR) imaging., Results: The TRAMP-C2 tumors grew rapidly to a predetermined endpoint size of 10 mm within 3 weeks, whereas the NEPC tumors grew at a slower rate over 7 weeks. The tumors were readily detected by MR and confidently identified when they were approximately 2-3 mm in size. The tumors were less well-defined on CT. The TRAMP-C2 tumors were characterized by amorphous sheets of poorly differentiated cells similar to a high-grade prostatic adenocarcinoma and frequent macroscopic peritoneal and lymph node metastases. In contrast, the NEPC's displayed a neuroendocrine morphology with polygonal cells arranged in nests and solid sheets and high count. There was a local invasion of the bladder and other adjacent tissues but no identifiable metastases. The TRAMP-C2 tumors were more hypoxic than the NEPC tumors., Conclusions: This novel preclinical orthotopic prostate cancer mouse model is suitable for either syngeneic or patient derived tumors and will be effective in developing and advancing the current selection of treatments for patients with prostate cancer., (© 2024 The Authors. The Prostate published by Wiley Periodicals LLC.)

High precision, image-guided radiotherapy (RT) has increased the therapeutic ratio, enabling higher tumor and lower normal tissue doses, leading to improved patient outcomes. Nevertheless, some patients remain at risk of developing serious side effects.In many clinical situations, the radiation tolerance of normal tissues close to the target volume limits the dose that can safely be delivered and thus the potential for tumor control and cure. This is particularly so in patients being re-treated for tumor progression or a second primary tumor within a previous irradiated volume, scenarios that are becoming more frequent in clinical practice.Various normal tissue 'radioprotective' drugs with the potential to reduce side effects have been studied previously. Unfortunately, most have failed to impact clinical practice because of lack of therapeutic efficacy, concern about concurrent tumor protection or excessive drug-related toxicity. This review highlights the evidence indicating that targeting the CXCL12/CXCR4 pathway can mitigate acute and late RT-induced injury and reduce treatment side effects in a manner that overcomes these previous translational challenges. Pre-clinical studies involving a broad range of normal tissues commonly affected in clinical practice, including skin, lung, the gastrointestinal tract and brain, have shown that CXCL12 signalling is upregulated by RT and attracts CXCR4-expressing inflammatory cells that exacerbate acute tissue injury and late fibrosis. These studies also provide convincing evidence that inhibition of CXCL12/CXCR4 signalling during or after RT can reduce or prevent RT side effects, warranting further evaluation in clinical studies. Greater dialogue with the pharmaceutical industry is needed to prioritize the development and availability of CXCL12/CXCR4 inhibitors for future RT studies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Tumor hypoxia is theorized to contribute to the aggressive biology of pancreatic ductal adenocarcinoma (PDAC). We previously reported that hypoxia correlated with rapid tumor growth and metastasis in patient-derived xenografts. Anticipating a prognostic relevance of hypoxia in patient tumors, we developed protocols for automated semi-quantitative image analysis to provide an objective, observer-independent measure of hypoxia. We further validated this method which can reproducibly estimate pimonidazole-detectable hypoxia in a high-through put manner., Methods: We studied the performance of three automated image analysis platforms in scoring pimonidazole-detectable hypoxia in resected PDAC (n = 10) in a cohort of patients enrolled in PIMO-PANC. Multiple stained tumor sections were analyzed on three independent image-analysis platforms, Aperio Genie (AG), Definiens Tissue Studio (TS), and Definiens Developer (DD), which comprised of a customized rule set., Results: The output from Aperio Genie (AG) had good concordance with manual scoring, but the workflow was resource-intensive and not suited for high-throughput analysis. TS analysis had high levels of variability related to misclassification of cells class, while the customized rule set of DD had a high level of reliability with an intraclass coefficient of more than 85%., Discussion: This work demonstrates the feasibility of developing a robust, high-performance pipeline for an automated, quantitative scoring of pimonidazole-detectable hypoxia in patient tumors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Siddiqui, Bilkey, McKee, Serra, Pintilie, Do, Xu, Tsao, Gallinger, Hill, Hedley and Dhani.)

Purpose. It has been postulated that the delivery of radiotherapy at ultra-high dose rates ('FLASH') reduces normal tissue toxicities by depleting them of oxygen. The fraction of normal tissue and cancer cells surviving radiotherapy depends on dose and oxygen levels in an exponential manner and even a very small fraction of tissue at low oxygen levels can determine radiotherapy response. To quantify the differential impact of FLASH radiotherapy on normal and tumour tissues, the spatial heterogeneity of oxygenation in tissue should thus be accounted for. Methods. The effect of FLASH on radiation-induced normal and tumour tissue cell killing was studied by simulating oxygen diffusion, metabolism, and radiolytic oxygen depletion (ROD) over domains with simulated capillary architectures. To study the impact of heterogeneity, two architectural models were used: (1) randomly distributed capillaries and (2) capillaries forming a regular square lattice array. The resulting oxygen partial pressure distribution histograms were used to simulate normal and tumour tissue cell survival using the linear quadratic model of cell survival, modified to incorporate oxygen-enhancement ratio effects. The ratio ('dose modifying factors') of conventional low-dose-rate dose and FLASH dose at iso-cell survival was computed and compared with empirical iso-toxicity dose ratios. Results. Tumour cell survival was found to be increased by FLASH as compared to conventional radiotherapy, with a 0-1 order of magnitude increase for expected levels of tumour hypoxia, depending on the relative magnitudes of ROD and tissue oxygen metabolism. Interestingly, for the random capillary model, the impact of FLASH on well-oxygenated (normal) tissues was found to be much greater, with an estimated increase in cell survival by up to 10 orders of magnitude, even though reductions in mean tissue partial pressure were modest, less than ∼7 mmHg for the parameter values studied. The dose modifying factor for normal tissues was found to lie in the range 1.2-1.7 for a representative value of normal tissue oxygen metabolic rate, consistent with preclinical iso-toxicity results. Conclusions. The presence of very small nearly hypoxic regions in otherwise well-perfused normal tissues with high mean oxygen levels resulted in a greater proportional sparing of normal tissue than tumour cells during FLASH irradiation, possibly explaining empirical normal tissue sparing and iso-tumour control results., (© 2022 Institute of Physics and Engineering in Medicine.)

Purpose: Cervical cancer is a global health problem. Despite the growth of prevention programs, there is an important need to improve the effectiveness of treatment for patients with invasive, locally advanced disease. In this study we examined (1) the efficacy of radiation therapy (RT) with cisplatin (RTCT) and an orally administered CXCR4 inhibitor suitable for clinical use, X4-136; (2) biomarkers of response to RTCT and X4-136; and (3) intestinal toxicity from RTCT and X4-136., Methods and Materials: Orthotopic cervical cancer xenografts derived from our patients were treated with RT (30 Gy; 2 Gy/d) and cisplatin (4 mg/kg/wk intraperitoneally) with or without concurrent X4-136 (100 mg/kg/d orally) for 3 weeks. Mice were euthanized immediately after treatment for biomarker assessment or followed to evaluate primary tumor growth delay and metastases. In separate experiments, acute and late intestinal injury were assessed histologically., Results: RTCT alone increased CXCL12/CXCR4 signaling, intratumoral accumulation of myeloid cells, and PD-L1 expression. The addition of X4-136 during RTCT abrogated these effects, improved primary tumor response, and reduced metastases. Furthermore, X4-136 increased the proportion of surviving intestinal crypt cells after irradiation, in keeping with a reduction in acute RT toxicity, and reduced late histologic changes of late RT toxicity., Conclusions: The combination of RTCT and the CXCR4 inhibitor X4-136 improves cervical cancer primary tumor control and reduces lymph node metastases, while also reducing normal tissue injury associated with adverse intestinal effects. Few if any pharmacologic strategies have expanded the therapeutic window with RT, suggesting that this combination warrants testing in clinical trials. These benefits might apply to other tumors where RTCT plays a curative role., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Hypoxia, the state of low oxygenation that often arises in solid tumours due to their high metabolism and irregular vasculature, is a major contributor to the resistance of tumours to radiation therapy (RT) and other treatments. Conventional RT extends treatment over several weeks or more, and nominally allows time for oxygen levels to increase ("reoxygenation") as cancer cells are killed by RT, mitigating the impact of hypoxia. Recent advances in RT have led to an increase in the use stereotactic body radiotherapy (SBRT), which delivers high doses in five or fewer fractions. For cancers such as pancreatic adenocarcinoma for which hypoxia varies significantly between patients, SBRT might not be optimal, depending on the extent to which reoxygenation occurs during its short duration. We used fluoro-5-deoxy-α-D-arabinofuranosyl)-2-nitroimidazole positron-emission tomography (FAZA-PET) imaging to quantify hypoxia before and after 5-fraction SBRT delivered to patient-derived pancreatic cancer xenografts orthotopically implanted in mice. An imaging technique using only the pre-treatment FAZA-PET scan and repeat dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) scans throughout treatment was able to predict the change in hypoxia. Our results support the further testing of this technique for imaging of reoxygenation in the clinic.

Background and Purpose: Gene expression signatures are often used to identify hypoxic tumors. However, intratumoral heterogeneity raises concern that multiple biopsies may be necessary to assess global hypoxia status. The objective of this study was to compare the impact of heterogeneity on the discriminative capacity of several previously described hypoxia gene signatures and determine if a single biopsy is sufficient to obtain a reliable estimate of hypoxia in cervical cancer., Materials and Methods: Multiple biopsies (33) were obtained from 11 locally advanced (FIGO IB to IVB) cervical cancers prior to treatment. Ten hypoxia gene signatures were analyzed. Variance component analysis was used to determine the ratio of within-tumor variability to total-tumor variability when one to five biopsies are available for analysis (W/T 1-5 ). The mean standardized error in the signature scores was estimated by comparing the score using one biopsy randomly selected from each tumor to the 'global' score using all available biopsies., Results: The ten hypoxia signatures were comprised of 6-99 genes each. The W/T 1 ratios for individual genes commonly found in the signatures ranged from 0.17 to 0.73. W/T 1 ratios for the signatures were generally lower (0.21-0.45), implying greater capacity to discriminate among tumors. With additional biopsies, the signature W/T ratios (ie W/T 2-5 ) decreased further. The mean error in the signature scores varied from 0.27 to 0.40 of one standard deviation, suggesting high capacity to discriminate among tumors with different global hypoxia scores., Conclusions: Compared with individual probes, hypoxia gene expression signatures are generally more consistent across multiple biopsies from different regions of a tumor and more tolerant of intratumoral heterogeneity., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2019 The Authors. Published by Elsevier B.V. on behalf of European Society for Radiotherapy and Oncology.)

Background: The CXCL12/CXCR4 chemokine pathway is involved in cervical cancer pathogenesis and radiation treatment (RT) response. We previously reported that radiochemotherapy (RTCT) and concurrent administration of the CXCR4 inhibitor plerixafor improved primary tumour response. The aims of this study were to determine optimal sequencing of RTCT and plerixafor, the mechanisms responsible for improved response and the effect of plerixafor on late intestinal toxicity., Methods: Orthotopic cervical cancer xenografts were treated with RTCT (30 Gy in 2 Gy fractions and cisplatin) with or without concurrent, adjuvant or continuous plerixafor. The endpoints were growth delay and molecular and immune cell changes at the end of treatment. Late intestinal toxicity was assessed by histologic examination of the rectum 90 days after a single 20 Gy fraction., Results: RTCT increased CXCL12/CXCR4 signalling and the intratumoral accumulation of myeloid cells; the addition of plerixafor mitigated these effects. All of the RTCT and plerixafor arms showed prolonged tumour growth delay compared to RTCT alone, with the adjuvant arm showing the greatest improvement. Plerixafor also reduced late intestinal toxicity., Conclusion: Adding Plerixafor to RTCT blunts treatment-induced increases in CXCL12/CXCR4 signalling, improves primary tumour response and reduces intestinal side effects. This combination warrants testing in future clinical trials.

Background and Purpose: Hypoxia, high interstitial fluid pressure (IFP) and immune effects have individually been shown to modulate radiotherapy (RT) response in cervical cancer. The aim of this study was to investigate the interplay between hypoxia or IFP and circulating neutrophil levels, and their combined effect on survival following RT., Material and Methods: A total of 287 FIGO stage IB to IIIB cervical cancer patients treated with RT or RT and cisplatin (RTCT) were included. Tumor hypoxia and IFP were measured at baseline prior to treatment. Absolute neutrophil count (ANC) was measured at baseline and weekly during treatment. Median follow up was 7.1 years., Results: High nadir ANC at the point of maximal myelosuppression was a stronger predictor of inferior survival than high baseline ANC after adjusting for clinical prognostic factors and treatment (RT vs. RTCT). The predictive effect of nadir ANC was most evident in patients with well-oxygenated tumors or tumors with high IFP at diagnosis., Conclusions: This study provides new information about the combined influence of the tumor microenvironment and myeloid cells on the survival of cervical cancer patients treated with RT/RTCT to motivate the development of new treatments based on molecular targeting of immune-based radioresistance pathways.

Given the distinct characteristics of low-energy electrons (LEEs), particularly at energies less than 30 eV, they can be applied to a wide range of therapeutic modalities to improve cancer treatment. LEEs have been shown to efficiently produce complex molecular damage resulting in substantial cellular toxicities. Since LEEs are produced in copious amounts from high-energy radiation beam, including photons, protons and ions; the control of LEE distribution can potentially enhance the therapeutic radio of such beams. LEEs can play a substantial role in the synergistic effect between radiation and chemotherapy, particularly halogenated and platinum-based anticancer drugs. Radiosensitizing entities containing atoms of high atomic number such as gold nanoparticles can be a source of LEE production if high-energy radiation interacts with them. This can provide a high local density of LEEs in a cell and produce cellular toxicity. Auger-electron-emitting radionuclides also create a high number of LEEs in each decay, which can induce lethal damage in a cell. Exploitation of LEEs in cancer treatment, however, faces a few challenges, such as dosimetry of LEEs and selective delivery of radiosensitizing and chemotherapeutic molecules close to cellular targets. This review first discusses the rationale for utilizing LEEs in cancer treatment by explaining their mechanism of action, describes theoretical and experimental studies at the molecular and cellular levels, then discusses strategies for achieving modification of the distribution and effectiveness of LEEs in cancerous tissue and their associated clinical benefit.

Background: The method of generating bioengineered skin constructs was pioneered several decades ago; nowadays these constructs are used regularly for the treatment of severe burns and nonhealing wounds. Commonly, these constructs are comprised of skin fibroblasts within a collagen scaffold, forming the skin dermis, and stratified keratinocytes overlying this, forming the skin epidermis. In the past decade there has been a surge of interest in bioengineered skins, with researchers seeking alternative cell sources, or scaffolds, from which constructs can be established, and for more biomimetic equivalents with skin appendages., Objectives: To evaluate whether human hair follicle dermal cells can act as an alternative cell source for engineering the dermal component of engineered skin constructs., Methods: We established in vitro skin constructs by incorporating into the collagenous dermal compartment: (i) primary interfollicular dermal fibroblasts, (ii) hair follicle dermal papilla cells or (iii) hair follicle dermal sheath cells. In vivo skins were established by mixing dermal cells and keratinocytes in chambers on top of immunologically compromised mice., Results: All fibroblast subtypes were capable of supporting growth of overlying epithelial cells, both in vitro and in vivo. However, we found hair follicle dermal sheath cells to be superior to fibroblasts in their capacity to influence the establishment of a basal lamina., Conclusions: Human hair follicle dermal cells can be readily interchanged with interfollicular fibroblasts and used as an alternative cell source for establishing the dermal component of engineered skin both in vitro and in vivo., (© 2016 British Association of Dermatologists.)

Purpose: There is an important need to improve the effectiveness of radio-chemotherapy (RTCT) for cervical cancer. The CXCL12/CXCR4 pathway can influence RT response by recruiting normal myeloid cells to the tumor microenvironment that in turn can exert radioprotective effects, and may promote metastases. The objective of this study was to explore the efficacy and toxicity of combining RTCT with CXCL12/CXCR4 inhibition in cervical cancer. Experimental Design: CXCR4 expression was measured in 115 patients with cervical cancer. Two primary orthotopic cervical cancer xenografts (OCICx) with different levels of CXCR4 expression were treated with RT (30 Gy: 15 daily fractions) and weekly cisplatin (4 mg/kg), with or without the CXCR4 inhibitor Plerixafor (5 mg/kg/day). The endpoints were tumor growth delay and lymph node metastases. Acute intestinal toxicity was assessed using a crypt cell assay. Results: There was a fivefold variation in CXCR4 mRNA expression in the patient samples, and good correlation between the expression in patients and in the xenografts. The combination of RTCT and Plerixafor produced substantial tumor growth delay and reduced lymph node metastases compared with RTCT alone in both of the xenograft models. There was a trend toward reduced acute intestinal toxicity with the addition of Plerixafor to RTCT. There were no changes in normal organ morphology to suggest increased late toxicity. Conclusions: This study demonstrates that the addition of Plerixafor to standard RTCT improves primary tumor response and reduces metastases in cervical cancer with no increase in toxicity. This combination warrants further investigation in phase I/II clinical trials. Clin Cancer Res; 23(5); 1242-9. ©2016 AACR ., (©2016 American Association for Cancer Research.)

Numerous studies have suggested that Cancer Initiating Cells (CIC) can be identified/enriched in cell populations obtained from solid tumors based on the expression of cell surface marker proteins. We used early passage primary cervix cancer xenografts to sort cells based on the expression of the intrinsic hypoxia marker Carbonic Anhydrase 9 (CA9) and tested their cancer initiation potential by limiting dilution assay. We demonstrated that CICs are significantly enriched in the CA9+ fraction in 5/6 models studied. Analyses of the expression of the stem cell markers Oct4, Notch1, Sca-1 & Bmi1 showed a trend toward an increase in the CA9+ populations, albeit not significant. We present evidence that enhanced autophagy does not play a role in the enhanced growth of the CA9+ cells. Our study suggests a direct in vivo functional link between hypoxic cells and CICs in primary cervix cancer xenografts.

Background: The Hedgehog (Hh) pathway is upregulated in cervical cancer and associated with poor outcome. We explored the effects of Hh pathway inhibition in combination with RTCT in a patient derived orthotopic cervical cancer xenograft model (OCICx)., Methods: 5E1, a monoclonal antibody for SHH, or Sonidegib (LDE225), a clinical SMO inhibitor (Novartis) were added to RTCT. We investigated tumour growth delay, metastasis and GI toxicity using orthotopic cervical cancer xenografts models. The xenografts were treated with radiotherapy (15 × 2 Gy daily fractions over 3 weeks) and weekly cisplatin 4 mg kg -1 concurrently, with or without 5E1 or Sonidegib (LDE225). The Hh inhibitors were administered by subcutaneous injection (5E1; 20 mg kg -1 weekly for 3 weeks), or by oral gavage (Sonidegib; 60 mg kg -1 daily for 3 weeks)., Results: We observed that both Hh inhibitors administered with RTCT were well tolerated and showed increased tumour growth delay, and reduced metastasis, with no increase in acute GI-toxicity relative to RTCT alone., Conclusions: Our data suggest Hh can be a valid therapeutic target in cervical cancer and supports data suggesting a potential therapeutic role for targeting Hh in patients undergoing RTCT. This warrants further investigation in clinical trials.

Tumours contain multiple different cell populations, including cells derived from the bone marrow as well as cancer-associated fibroblasts and various stromal populations including the vasculature. The microenvironment of the tumour cells plays a significant role in the response of the tumour to radiation treatment. Low levels of oxygen (hypoxia) caused by the poorly organized vasculature in tumours have long been known to affect radiation response; however, other aspects of the microenvironment may also play important roles. This article reviews some of the old literature concerning tumour response to irradiation and relates this to current concepts about the role of the tumour microenvironment in tumour response to radiation treatment. Included in the discussion are the role of cancer stem cells, radiation damage to the vasculature and the potential for radiation to enhance immune activity against tumour cells. Radiation treatment can cause a significant influx of bone marrow-derived cell populations into both normal tissues and tumours. Potential roles of such cells may include enhancing vascular recovery as well as modulating immune reactivity.

Previous reports have suggested that the hypoxic microenvironment provides a niche that supports tumor stem cells, and that this might explain clinical observations linking hypoxia to metastasis. To test this, we examined the effects of a hypoxia-activated prodrug, TH-302, on the tumor-initiating cell (TIC) frequency of patient-derived pancreatic xenografts (PDX).The frequencies of TIC, measured by limiting dilution assay, varied widely in 11 PDX models, and were correlated with rapid growth but not with the levels of hypoxia. Treatment with either TH-302 or ionizing radiation (IR), to target hypoxic and well-oxygenated regions, respectively, reduced TIC frequency, and the combination of TH-302 and IR was much more effective in all models tested. The combination was also more effective than TH-302 or IR alone controlling tumor growth, particularly treating the more rapidly-growing/hypoxic models. These findings support the clinical utility of hypoxia targeting in combination with radiotherapy to treat pancreatic cancers, but do not provide strong evidence for a hypoxic stem cell niche., Competing Interests: The authors have no conflict of interest to report.

Enhanced autophagy has been observed in hypoxic regions of solid tumors. Here we address the hypothesis that autophagy is required for survival of hypoxic cells. We evaluated sensitivity to hypoxia of three human tumor cell lines (MCF7, PC3, and LNCaP) and their autophagy-deficient variants with shRNA knockdown of the genes ATG7 and BECLIN1. Hypoxia-induced cell death was more rapid for autophagy-deficient cells and was increased in the presence of the proton pump inhibitor pantoprazole that inhibits autophagy. Autophagy-deficient cells had a lower rate of oxygen consumption than wild-type cells. In xenografts derived from the three cell lines, autophagy (as determined by increased LC3 and reduced p62/SQSTM1) colocalized with hypoxic regions (identified by EF5). Xenografts derived from autophagy-deficient cells grew more slowly than wild-type tumors. Both LC3 expression and hypoxia were decreased in xenografts generated from single-knockdown cells and absent in double-knockdown tumors. Our results are consistent with the hypothesis that autophagy facilitates the survival of hypoxic cells, although reduced oxygen consumption of autophagy-deficient cells may contribute to lack of hypoxia in tumors derived from them. Because hypoxia is associated with resistance to anticancer therapy, inhibition of autophagy has potential to enhance the effectiveness of cancer treatment., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: Preclinical studies have shown that angiogenesis inhibition can improve response to radiation therapy (RT). The purpose of this phase 1 study was to examine the angiogenesis inhibitor sorafenib in patients with cervical cancer receiving radical RT and concurrent cisplatin (RTCT)., Methods and Materials: Thirteen patients with stage IB to IIIB cervical cancer participated. Sorafenib was administered daily for 7 days before the start of standard RTCT in patients with early-stage, low-risk disease and also during RTCT in patients with high-risk disease. Biomarkers of tumor vascularity, perfusion, and hypoxia were measured at baseline and again after 7 days of sorafenib alone before the start of RTCT. The median follow-up time was 4.5 years., Results: Initial complete response was seen in 12 patients. One patient died without achieving disease control, and 4 experienced recurrent disease. One patient with an extensive, infiltrative tumor experienced pelvic fistulas during treatment. The 4-year actuarial survival was 85%. Late grade 3 gastrointestinal toxicity developed in 4 patients. Sorafenib alone produced a reduction in tumor perfusion/permeability and an increase in hypoxia, which resulted in early closure of the study., Conclusions: Sorafenib increased tumor hypoxia, raising concern that it might impair rather than improve disease control when added to RTCT., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The cancer stem cell model in solid tumors has evolved significantly from the early paradigm shifting work highlighting parallels between the stem cell hierarchy in hematologic malignancies and solid tumors. Putative stem cells can dedifferentiated, be induced by context, and be the result of accumulated genetic mutations. The simple hypothesis that stem cell therapies will overcome the minority of cells that lead to recurrence has evolved with it. Nevertheless, the body of evidence that this field is clinically relevant in patients and patient care has grown with the complexity of the hypotheses, and numerous clinical strategies to target these cells have been identified. Herein we review this progress and highlight the work still outstanding.

Recent modifications in the metrics for reimbursement have reinforced the importance of radiology service-delivery experiences of patients. Evaluating current radiology practices calls for reflection on the various touch points with patients, as well as their overall satisfaction. If problems occur during encounters, service failure, or lack of satisfactory medical experiences can be transformed through service recovery, whereby patients-as-customers are given chances to voice their concerns, and health care providers across the spectrum can work together to resolve problematic issues. This paper takes a systemic view of the patient experience as embedded in the care continuum, recognizing that different beliefs, attitudes, and behaviors of members of the health care team can negatively affect or sabotage patient satisfaction. Although radiologists are only one of many roles in the care continuum, recommendations are discussed for how they can integrate service satisfaction as a pervasive communal goal among all health care team members., (Copyright © 2015 American College of Radiology. Published by Elsevier Inc. All rights reserved.)

The results from many studies indicate that most solid tumors, regardless of site of origin, contain hypoxic regions. Experimental studies have demonstrated that, apart from the well-known protective effect of hypoxia on the radiation response of cells and tissues, hypoxic conditions can also result in modified gene expression patterns, causing (to a greater or lesser extent in different cell populations) genomic instability, increased invasive capacity, higher propensity to metastasize, enhanced stem cell properties, and ability to survive nutrient deprivation. Clinical trials of hypoxia-targeted treatments have demonstrated improved local tumor control and patient survival in a number of tumor sites. However, our improved understanding of the underlying biology of cellular responses to hypoxia, and its potential interactions with the heterogeneous nature of tumor phenotypes, makes it likely that not every tumor that contains regions of hypoxia would necessarily need (or benefit from) such treatments. New more effective treatments are emerging, but it is likely that these treatments would have the biggest clinical effect in situations where tumor hypoxia is a primary driver of cancer behavior. The challenge for the Radiation Oncology community is the development of robust precision cancer medicine strategies for identifying patients with such tumors, in the setting of other etiological, genomic, and host-tumor factors, and treating these patients with the appropriate hypoxia-targeting strategy to reduce the effect of hypoxia on radiation treatment response. In this context, it is important to consider not only the hypoxic state of the tumor at diagnosis but also the changing characteristics of this state during the course of treatment., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Hypoxia is thought to be an adverse feature of pancreatic cancer, but direct measurement in patients is technically challenging. To address this, we characterised the intra/interpatient heterogeneity of hypoxia in surgical specimens from patients who received the 2-nitroimidazole tracer pimonidazole pre-operatively., Methods: Pimondazole was given intravenously 16-20 h before pancreatectomy, and the extent and intratumoral heterogeneity of hypoxia determined by image analysis applied to multiple tissue blocks stained by immunohistochemistry. Intra/interpatient heterogeneity was estimated by variance component analysis., Results: Pimonidazole staining was analysed in 10 tumours. The extent of labelling varied amongst patients (0-26%), with a broader range of hypoxia in the epithelial (1-39%) compared with the stromal (1-13%) compartments. Variance component analysis demonstrated greater inter- than intrapatient variability of hypoxia, and that multiple (4-5) tumour sections are required to provide a consistent evaluation of its extent in individual tumours., Conclusions: There is significant intra- and intertumoral heterogeneity of hypoxia in pancreatic cancers, and these do not appear to be generally more hypoxic than other cancer types. This study establishes the feasibility to assess hypoxia in pancreatic cancer patients using pimonidazole, but questions the reliability of measurements made using a single tissue section.

Solid tumors contain regions of chronic and cyclic hypoxia. Chronic hypoxia can downregulate RAD51 and sensitize cells to PARP inhibition. Herein, we show that RAD51 expression, cell survival and toxicity to PARP inhibition is not affected under cyclic hypoxic conditions. This suggests that PARP inhibition may be selectively toxic in tumor sub-regions associated with chronic hypoxia., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Background: Cervical cancer is the third most common cancer in women globally, and despite treatment, distant metastasis and nodal recurrence will still develop in approximately 30% of patients. The ability to predict which patients are likely to experience distant relapse would allow clinicians to better tailor treatment. Previous studies have investigated the role of chromosomal instability (CIN) in cancer, which can promote tumour initiation and growth; a hallmark of human malignancies. In this study, we sought to examine the published CIN70 gene signature in a cohort of cervical cancer patients treated at the Princess Margaret (PM) Cancer Centre and an independent cohort of The Cancer Genome Atlas (TCGA) cervical cancer patients, to determine if this CIN signature associated with patient outcome., Methods: Cervical cancer samples were collected from 79 patients, treated between 2000-2007 at the PM, prior to undergoing curative chemo-radiation. Total RNA was extracted from each patient sample and analyzed using the GeneChip Human Genome U133 Plus 2.0 array (Affymetrix)., Results: High CIN70 scores were significantly related to increased chromosomal alterations in TCGA cervical cancer patients, including a higher percentage of genome altered and a higher number of copy number alterations. In addition, this same CIN70 signature was shown to be predictive of para-aortic nodal relapse in the PM Cancer Centre cohort., Conclusions: These findings demonstrate that chromosomal instability plays an important role in cervical cancer, and is significantly associated with patient outcome. For the first time, this CIN70 gene signature provided prognostic value for patients with cervical cancer.

Cervical cancer remains the third most frequently diagnosed and fourth leading cause of cancer death in women worldwide. We sought to develop a micro-RNA signature that was prognostic for disease-free survival, which could potentially allow tailoring of treatment for cervical cancer patients. A candidate prognostic 9-micro-RNA signature set was identified in the training set of 79 frozen specimens. However, three different approaches to validate this signature in an independent cohort of 87 patients with formalin-fixed paraffin-embedded (FFPE) specimens, were unsuccessful. There are several challenges and considerations associated with developing a prognostic micro-RNA signature for cervical cancer, namely: tumour heterogeneity, lack of concordance between frozen and FFPE specimens, and platform selection for global micro-RNA expression profiling in this disease. Our observations provide an important cautionary tale for future miRNA signature studies for cervical cancer, which can also be potentially applicable to miRNA profiling studies involving other types of human malignancies.

Although of fundamental importance to the treatment of cancer patients, the quantitative study of drug distribution and action in vivo at the single cell level is challenging. We used the recently-developed technique of mass cytometry to measure cisplatin uptake into individual tumor cells (Pt atoms/cell), combined with measurement of the rate of IdU incorporation into DNA (I(127) atoms/cell/min) and tumor hypoxia identified by the 2-nitroimidazole EF5 in cisplatin-treated BxPC-3 and ME-180 xenografts. Pt levels of 10(5) to 10(6) atoms/cell were obtained following a single cisplatin treatment using clinically relevant doses. Cisplatin caused cell cycle arrest in a dose- and time-dependent manner that paralleled effects in vitro, and it readily penetrated into hypoxic tumor regions. Similar levels of Pt/cell were found in xenografts treated with oxaliplatin. Mass cytometry offers the unique capability to study the cellular uptake and anticancer effects of platinum-containing drugs at the single cell level in animal models, and it has the potential for application to samples obtained from cancer patients during treatment., (© 2014 UICC.)

Cervical cancer is the third most common cancer in women worldwide. In the present study, global microRNA profiling for 79 cervical cancer patient samples led to the identification of miR-218 down-regulation in cervical cancer tissues compared to normal cervical tissues. Lower miR-218 expression was associated significantly with worse overall survival (OS), disease-free survival (DFS), and pelvic/aortic lymph node recurrence. In vitro, miR-218 over-expression decreased clonogenicity, migration, and invasion. Survivin (BIRC5) was subsequently identified as an important cervical cancer target of miR-218 using in silico prediction, mRNA profiling, and quantitative real-time PCR (qRT-PCR). Concordant with miR-218 over-expression, survivin knockdown by siRNA decreased clonogenicity, migration, and invasion. YM155, a small molecule survivin inhibitor, significantly suppressed tumor growth and lymph node metastasis in vivo. Our findings demonstrate that the miR-218~survivin axis inhibits cervical cancer progression by regulating clonogenicity, migration, and invasion, and suggest that the inhibition of survivin could be a potential therapeutic strategy to improve outcome in this disease.

Standard treatment for women who are diagnosed with stage IIB through IVA cervical cancer consists of cisplatin-based chemotherapy and radiation. Current options for patients with recurrent and metastatic disease are limited, and their median overall survival is <12 months. To date, biologic therapy has had little impact on survival, so identification of potential new targets is urgently required to develop novel therapeutic strategies. Developing relevant animal models for human cervix cancer is important to further enhance our understanding of the characteristics of these tumors and for identification and assessment of novel therapies. We have established a panel of orthotopically passaged xenografts (OCICx) by implanting cervix tumor pieces from patient biopsies directly into the cervix of mice. The tumors have been passaged up to five generations, were characterized histologically for tumor and stromal content and, where possible, related to similar measurements in the original patient biopsy. The tumors were found to metastasize to the para-aortic lymphnodes allowing assessment of their metastatic potential. Preliminary studies demonstrate aberrant expression of genes in the Hedgehog (Hh) pathway in the xenografts similar to findings in primary cervix cancers. The OCICx xenografts represent unique models to test strategies for targeting essential pathways in cervix cancer and metastasis.

Radiotherapy (RT) with concurrent cisplatin (CRT) is standard treatment for locally advanced cervical cancer. However, not all patients benefit from the addition of cisplatin to RT alone. This study explored the value of pretreatment tumor interstitial fluid pressure (IFP) and hypoxia measurements as predictors of cisplatin response in 291 patients who were treated with RT (1994-1998) or RT plus concurrent cisplatin (1999-2009). Clinical characteristics were similar between the two groups, apart from a greater proportion of patients with pelvic lymph node metastases and hypoxic tumors in the CRT cohort. Patients were followed for a median duration of 5.6 years. Information about recurrence and survival was recorded prospectively. The addition of cisplatin to RT improved survival compared to treatment with RT alone (HR 0.61, p = 0.0097). This improvement was confined to patients with high-IFP tumors at diagnosis (HR 0.40, p = 0.00091). There was no benefit of adding cisplatin in those with low-IFP tumors (HR 1.05, p = 0.87). There was no difference in the effectiveness of cisplatin in patients with more or less hypoxic tumors. In conclusion, patients with locally advanced cervical cancer and high tumor IFP at diagnosis have greater benefit from the addition of cisplatin to RT than those with low IFP. This may reflect high tumor cell proliferation, which is known to influence IFP, local tumor control and patient survival., (© 2013 UICC.)

Background: Tumor initiation, growth and relapse after therapy are thought to be driven by a population of cells with stem cell characteristics, named cancer stem cells (CSC). The regulation of their radiation resistance and their maintenance is poorly understood. CSC are believed to reside preferentially in special microenvironmental niches located within tumor tissues. The features of these niches are of crucial importance for CSC self-renewal, metastatic potential and therapy resistance. One of the characteristics of solid tumors is occurrence of less oxygenated (hypoxic regions), which are believed to serve as so-called hypoxic niches for CSC., Purpose: The purpose of this review was the critical discussion of the supportive role of hypoxia and hypoxia-related pathways during cancer progression and radiotherapy resistance and the relevance for therapeutic implications in the clinic., Conclusion: It is generally known since decades that hypoxia inside solid tumors impedes chemo- and radiotherapy. However, there is limited evidence to date that targeting hypoxic regions during conventional therapy is effective. Nonetheless improved hypoxia-imaging technologies and image guided individualized hypoxia targeted therapy in conjunction with the development of novel molecular targets may be able to challenge the protective effect on the tumor provided by hypoxia.

Glioblastoma multiforme (GBM) is characterized by a pathogenic vasculature that drives aggressive local invasion. Recent work suggests that GBM cells recruit bone marrow-derived progenitor cells (BMDC) to facilitate recurrence after radiotherapy, but how this may be achieved is unclear. In this study, we established the spatiotemporal and regional contributions of perivascular BMDCs (pBMDC) to GBM development. We found an increased recruitment of BMDCs to GBM in response to tumor growth and following radiotherapy. However, in this study, BMDCs did not differentiate into endothelial cells directly but rather provided a perivascular support role. The pBMDCs were shown to associate with tumor vasculature in a highly region-dependent manner, with central vasculature requiring minimal pBMDC support. Region-dependent association of pBMDC was regulated by VEGF. In the absence of VEGF, following radiotherapy or antiangiogenic therapy, we documented an increase in Ang2 that regulated recruitment of pBMDCs to maintain the vulnerable central vasculature. Together, our results strongly suggested that targeting pBMDC influx along with radiation or antiangiogenic therapy would be critical to prevent vascular recurrence of GBM., (©2014 American Association for Cancer Research.)

Purpose: To investigate the outcome of suppression of the renin angiotensin system using captopril combined with an antioxidant (Eukarion [EUK]-207) for mitigation of radiation-induced lung damage in rats., Methods and Materials: The thoracic cavity of female Sprague-Dawley rats was irradiated with a single dose of 11 Gy. Treatment with captopril at a dose of 40 mg/kg/d in drinking water and EUK-207 given by subcutaneous injection (8 mg/kg daily) was started 1 week after irradiation (PI) and continuing until 14 weeks PI. Breathing rate was monitored until the rats were killed at 32 weeks PI, when lung fibrosis was assessed by lung hydroxyproline content. Lung levels of the cytokine transforming growth factor-β1 and macrophage activation were analyzed by immunohistochemistry. Oxidative DNA damage was assessed by 8-hydroxy-2-deoxyguanosine levels, and lipid peroxidation was measured by a T-BARS assay., Results: The increase in breathing rate in the irradiated rats was significantly reduced by the drug treatments. The drug treatment also significantly decreased the hydroxyproline content, 8-hydroxy-2-deoxyguanosine and malondialdehyde levels, and levels of activated macrophages and the cytokine transforming growth factor-β1 at 32 weeks. Almost complete mitigation of these radiation effects was observed by combining captopril and EUK-207., Conclusion: Captopril and EUK-207 can provide mitigation of radiation-induced lung damage out to at least 32 weeks PI after treatment given 1-14 weeks PI. Overall the combination of captopril and EUK-207 was more effective than the individual drugs used alone., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Conditions of poor oxygenation (hypoxia) are present in the majority of solid human tumors and are associated with poor patient prognosis due to both hypoxia-mediated resistance to treatment, and to hypoxia induced biological changes that promote increased malignancy, including metastasis. Tumor cells respond to hypoxia by activating several oxygen-sensitive signaling pathways that include the hypoxia inducible factor 1/2 (HIF1/2) signalling pathways and the unfolded protein response (UPR), which alter gene expression to promote adaptation and survival during hypoxic conditions. Furthermore, these hypoxia responsive pathways can lead to changes in gene expression and cellular phenotype that influence the potential of cancer cells to metastasize. However, the hypoxia-induced signaling events that promote tumor metastasis are still relatively poorly understood. Previous studies have largely focused on the contribution of the HIF signaling pathway to hypoxia-mediated metastasis. However, recent evidence demonstrates that hypoxic activation of the UPR is also an important mediator of metastasis.

Numerous studies have demonstrated the presence of cancer stem cells (CSCs) within solid tumors. Although the precursor of these cells is not clearly established, recent studies suggest that the phenotype of CSCs may be quite plastic and associated with the epithelial-to-mesenchymal transition (EMT). In patients, the presence of EMT and CSCs has been implicated in increased resistance to radiotherapy. Hypoxia, a negative prognostic factor for treatment success, is a potent driver of a multitude of molecular signalling pathways that allow cells to survive and thrive in the hostile tumor microenvironment and can induce EMT. Hypoxia also provides tumor cells with cues for maintenance of a stem-like state and may help to drive the linkage between EMT and CSCs. Understanding the biology of CSCs, the EMT phenotype and their implications in therapeutic relapse may provide crucial new approaches in the development of improved therapeutic strategies., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Purpose: Conditions of poor oxygenation (hypoxia) are present in many human tumors, including cervix cancer, and are associated with increased risk of metastasis and poor prognosis. Hypoxia is a potent activator of the PERK/eIF2α signaling pathway, a component of the unfolded protein response (UPR) and an important mediator of hypoxia tolerance and tumor growth. Here, the importance of this pathway in the metastasis of human cervix carcinoma was investigated., Experimental Design: Amplification and expression of LAMP3, a UPR metastasis-associated gene, was examined using FISH and immunofluorescence in a cohort of human cervix tumors from patients who had received oxygen needle electrode tumor oxygenation measurements. To evaluate the importance of this pathway in metastasis in vivo, we constructed a series of inducible cell lines to interfere with PERK signaling during hypoxia and used these in an orthotopic cervix cancer model of hypoxia-driven metastasis., Results: We show that LAMP3 expression in human cervix tumors is augmented both by gene copy number alterations and by hypoxia. Induced disruption of PERK signaling in established orthotopic xenografts resulted in complete inhibition of hypoxia-induced metastasis to the lymph nodes. This is due, in part, to a direct influence of the UPR pathway on hypoxia tolerance. However, we also find that LAMP3 is a key mediator of hypoxia-driven nodal metastasis, through its ability to promote metastatic properties including cell migration., Conclusion: These data suggest that the association between hypoxia, metastasis, and poor prognosis is due, in part, to hypoxic activation of the UPR and expression of LAMP3. Clin Cancer Res; 19(22); 6126-37. ©2013 AACR.

To test if proteolysis is involved in tumor cell extravasation, we developed an in vitro model where tumor cells cross an endothelial monolayer cultured on a basement membrane. Using this model we classified the ability of the cells to transmigrate through the endothelial cell barrier onto the underlying matrix, and scored this invasion according to the stage of passage through the endothelium. Metalloproteinase inhibitors reduced tumor cell extravasation by at least 35%. Visualization of protease and cell adhesion molecules by confocal microscopy demonstrated the cell surface localization of MMP-2, MMP-9, MT1-MMP, furin, CD44 and αvβ3, during the process of transendothelial migration. By the addition of inhibitors and bio-modulators we assessed the functional requirement of the aforementioned molecules for efficient migration. Proteolytic digestion occurred at the cell-matrix interface and was most evident during the migratory stage. All of the inhibitors and biomodulators affected the transition of the tumor cells into the migratory stage, highlighting the most prevalent use of proteolysis at this particular step of tumor cell extravasation. These data suggest that a proteolytic interface operates at the tumor cell surface within the tumor-endothelial cell microenvironment.