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Purpose Cervical cancer represents the fourth most frequent malignancy in the world among women, and mortality has remained stable for the past 4 decades. Intravenous cisplatin with concurrent radiation therapy is the standard-of-care for patients with local and regional cervical cancer. However, cisplatin induces serious dose-limiting systemic toxicities and recurrence frequently occurs. In this study, we aimed to develop an intracervical drug delivery system that allows cisplatin release directly into the tumor and minimize systemic side effects. Methods and Materials Twenty patient biopsies and 5 cell lines treated with cisplatin were analyzed for platinum content using inductively coupled plasma mass spectrometry. Polymeric implants loaded with cisplatin were developed and evaluated for degradation and drug release. The effect of local or systemic cisplatin delivery on drug biodistribution as well as tumor burden were evaluated in vivo, in combination with radiation therapy. Results Platinum levels in patient biopsies were 6-fold lower than the levels needed for efficacy and radiosensitization in vitro. Cisplatin local delivery implant remarkably improved drug specificity to the tumor and significantly decreased accumulation in the blood, kidney, and other distant normal organs, compared with traditional systemic delivery. The localized treatment further resulted in complete inhibition of tumor growth. Conclusions The current standard-of-care systemic administration of cisplatin provides a subtherapeutic dose. We developed a polymeric drug delivery system that delivered high doses of cisplatin directly into the cervical tumor, while lowering drug accumulation and consequent side effects in normal tissues. Moving forward, these data will be used as the basis of a future first-in-human clinical trial to test the efficacy of localized cisplatin as adjuvant or neoadjuvant chemotherapy in local and regional cervical cancer.

The synthesis of d-glucoheptose derivative containing a boronic moiety is described herein. Starting from benzyl 6,7-dideoxy-2,3,4-tri-O-benzyl-β-d-gluco-ept-6-enopyranoside, the introduction of the boronic acid was performed through a metathesis reaction by using MIDA vinyl boronic acid and the 2nd generation Grubbs catalyst. Hydrogenation led to the final product in only two reaction steps. This new sugar-containing boronic acid in the skeleton could mimic carbohydrate behavior and follow the glucose uptake in living cells. The in vitro toxicity tests performed in fibroblasts and glioma tumor cell lines showed minimal toxicity. Boron uptake measured using ICP-MS was minimal in fibroblasts, while in glioma cells showed a value of 6 ng of total boron accumulation per mg of cells, implying that compound 1a is able to accumulate selectively in the tumor tissues compared to normal.

Background CD138 has been the gold-standard surface marker to detect multiple myeloma (MM) cells for decades; however, drug resistant minimal-residual disease (MRD) and circulating tumor cells (CTCs) were shown to have lower expression of this marker. We previously published that residual MM cells following treatment in vivo were hypoxic and the combination of hypoxia and chemotherapy such as bortezomib downregulated CD138 expression, thereby making this marker unsuitable for MM detection. Needless to say, accurate number of MM cells is critical in diagnosis, autologous transplantation, MRD and drug efficiency assessment. Moreover, CTCs are considered an unfavorable prognostic factor and indicate an aggressive form of the disease, and therefore detecting CTCs can be used as a powerful prognostic tool for MM. Methods We used an alternative biomarker-set using flow cytometry defining MM cells as any cell that expresses CD38 but excluding T cell (CD3), B cell (CD19), NK cell (CD16), monocyte/macrophage (CD14), neutrophil/eosinophil (CD16), and basophil/dendritic cell (CD123). We demonstrated previously that this approach it widely available due to accessibility of flow cytometry, inexpensive, and works independently of hypoxic-, CD138 expression- and treatment status. We analyzed primary patient samples (n=50) with complete response or very good partial response for MRD and CTCs and correlated the numbers of detected MM with patients’ time-to-progression (TTP) obtained from a clinical data base. Results We found that the alternative biomarker-set identifies MM cells more precisely and at higher numbers than CD138 marker by flow or histology. Moreover, we found a correlation between the number of MM cells detected and TTP in these patients: the amount of MM cells detected by the new method ranged between 0.5-7.3%, and patients who progressed sooner than two years had 2.5-fold higher percentage of MM cells compared to patients who relapsed later than 2 years. Similarly, patients who relapsed sooner than 3 years had 4-fold higher number of MM cells than patients who relapsed later than 3 years. We further found that, among all patients who had more than 2% of MM cells detected by the new method had a mean TPP of about 20 months, while patients who had less than 2% had a mean TPP of about 38 months. Testing the prevalence of CTCs in MM patients with progressive disease using CD138 or the new method, demonstrated that CD138 detected minimal amounts of MM cells in all patients (less than 0.1%), while the new method detected a range between 0.1 - 1.8% of MM cells in the peripheral blood. Conclusion These results suggest that the alternative biomarker-set detected MM cells which correlated with relapse in MM patients. Therefore, the amount of residual cells in the bone marrow and circulating myeloma cells can be used as a prognostic marker in MM patients. Further examination to characterize this population and its role in MM relapse is warranted.

Background Multiple myeloma (MM) is a cancer of plasma cells within the bone marrow (BM) and remains to be incurable. Tumor-associated macrophages (TAMs) are a major immunosuppressive component in the tumor microenvironment and display the pro-tumor M2 phenotype, supporting tumor proliferation, survival, and drug resistance. Targeting TAMs to block their pro-tumor functions represent a promising class of cancer immunotherapy. IL10 is a key immunosuppressive cytokine that leads to recruitment and development of TAMs. In this study, we investigate the role of IL10 in MM TAM development, and hypothesize that inhibition of IL10/IL10R signaling in TAMs will reprogram them for MM killing and overcome drug resistance. Methods Macrophage (MPP) polarization to M1 (CD80+) or M2 (CD163+) is determined by antibody staining and flow cytometry. In vitro and ex vivo studies were carried out in our lab’s proprietary 3D tissue-engineered BM model (3DTEBM). First, we compared TAM M2/M1 ratio as well as IL10 cytokine levels in the BM of healthy and MM subjects. To study the role of IL10 in TAM development, we co-cultured MΦ with increasing MM cells or rhIL10 in the 3DTEBM for 3 days and analyzed the resulting M2/M1 ratio. To determine the effect of IL10R inhibition, MM and MΦ co-cultures, or unsorted patient BM were treated with or without 5ug/ml α-IL10R monoclonal antibody (mAb), and TAM phenotype was analyzed after 3 days. Additionally, humanized huCD34-NCG mice were inoculated with MM cells and treated with α-IL10R mAb 3 times/week for 2 weeks; mice femurs were flushed and BM cells were analyzed for TAM phenotype. Finally, we investigated α-IL10R mAb as combination treatment with chemotherapy in MM-GFP and MΦ co-cultures; MM survival was determined by count of GFP+ cells and apoptosis was determined by Annexin/PI staining with flow cytometry. Results Compared to healthy subjects, M2/M1 ratio in MM patient TAMs were 3-fold higher, and BM IL10 level was 3.8-fold higher. Additionally, increasing MM cell ratio in co-cultures induced TAM polarization to M2, and was contributed by IL10. Importantly, α-IL10R mAb was able to reverse MM induced M2 TAM phenotype in vitro, ex vivo, and in vivo. Moreover, the presence of TAMs resulted in significant MM proliferation and chemotherapy resistance toward lenalidomide and dexamethasone. However, combination treatment with α-IL10R mAb completely reversed the drug resistance and abrogated cancer cells in vitro, partially due to induction of apoptosis in MM cells. Conclusions In summary, we have shown that MM induced M2 polarization of TAMs in an IL-10 dependent fashion, and disruption of IL10/IL10R signaling reversed M2 phenotype in TAMs, and overcame TAM-supported drug resistance. Future studies are warranted to examine mechanism behind overcoming drug resistance as well as the effect of α-IL10R immunotherapy in vivo and in patients.

Vascular endothelial cells (ECs) are ideal gene therapy targets as they provide widespread tissue access and are the first contact surfaces following intravenous vector administration. Human recombinant adenovirus serotype 5 (Ad5) is the most frequently used gene transfer system because of its appreciable transgene payload capacity and lack of somatic mutation risk. However, standard Ad5 vectors predominantly transduce liver but not the vasculature following intravenous administration. We recently developed an Ad5 vector with a myeloid cell-binding peptide (MBP) incorporated into the knob-deleted, T4 fibritin chimeric fiber (Ad.MBP). This vector was shown to transduce pulmonary ECs presumably via a vector handoff mechanism. Here we tested the body-wide tropism of the Ad.MBP vector, its myeloid cell necessity, and vector-EC expression dose response. Using comprehensive multi-organ co-immunofluorescence analysis, we discovered that Ad.MBP produced widespread EC transduction in the lung, heart, kidney, skeletal muscle, pancreas, small bowel, and brain. Surprisingly, Ad.MBP retained hepatocyte tropism albeit at a reduced frequency compared with the standard Ad5. While binding specifically to myeloid cells ex vivo, multi-organ Ad.MBP expression was not dependent on circulating monocytes or macrophages. Ad.MBP dose de-escalation maintained full lung-targeting capacity but drastically reduced transgene expression in other organs. Swapping the EC-specific ROBO4 for the CMV promoter/enhancer abrogated hepatocyte expression but also reduced gene expression in other organs. Collectively, our multilevel targeting strategy could enable therapeutic biological production in previously inaccessible organs that pertain to the most debilitating or lethal human diseases.