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Introduction: Effective infiltration of chimeric antigen receptor T (CAR-T) cells into solid tumors is critical for achieving a robust antitumor response and improving therapeutic outcomes. While CAR-T cell therapies have succeeded in hematologic malignancies, their efficacy in solid tumors remains limited due to poor tumor penetration and an immunosuppressive tumor microenvironment. This study aimed to evaluate the potential of low-dose radiotherapy (LDRT) administered before T-cell therapy to enhance the antitumor effect by promoting CAR-T cell infiltration. We hypothesized that combining LDRT with T-cell therapy would improve tumor control and survival compared to either treatment alone. Methods: We investigated this hypothesis using two NSG mouse models bearing GSU or CAPAN-2 solid tumors. The mice were treated with engineered CAR-T cells targeting guanyl cyclase-C (GCC) or mesothelin as monotherapy or in combination with LDRT. Additionally, we extended this approach to a C57BL/6 mouse model implanted with MC38-gp100+ cells, followed by adoptive transfer of pmel+ T cells before and after LDRT. Tumor growth and survival outcomes were monitored in all models. Furthermore, we employed atomic force microscopy (AFM) in a small cohort to assess the effects of radiotherapy on tumor stiffness and plasticity, exploring the role of tumor nanomechanics as a potential biomarker for treatment efficacy. Results: Our results demonstrated enhanced tumor control and prolonged survival in mice treated with LDRT followed by T-cell therapy across all models. The combination of LDRT with CAR-T or pmel+ T-cell therapy led to superior tumor suppression and survival compared to monotherapy, highlighting the synergistic impact of the combined approach. Additionally, AFM analysis revealed significant changes in tumor stiffness and plasticity in response to LDRT, suggesting that the nanomechanical properties of the tumor may be predictive of therapeutic response. Discussion: The findings of this study highlight the transformative potential of incorporating LDRT as a precursor to adoptive T-cell therapy in solid tumors. By promoting CAR-T and pmel+ T-cell infiltration into the tumor microenvironment, LDRT enhanced tumor control and improved survival outcomes, offering a promising strategy to overcome the challenges associated with CAR-T therapy in solid tumors. Additionally, the changes in tumor nanomechanics observed through AFM suggest that tumor stiffness and plasticity could be biomarkers for predicting treatment outcomes. These results support further investigation into the clinical application of this combined approach to improve the efficacy of cellbased therapies in patients with solid tumors. [ABSTRACT FROM AUTHOR]

Simple Summary: Penile cancer is a rare but aggressive cancer. After it metastasizes, the median survival time is less than 12 months. The overall response rate to common first-line combination chemotherapy treatments is approximately 50%. There is an urgent need in advanced-penile-cancer treatment to find novel therapies that would generate better response rates than standard chemotherapy thus far and have less toxicity. Partially due to its rarity, there are few animal models and cell lines of penile cancer. We report on the generation of seven penile cancer animal models that were created by directly implanting human tumor tissue into immunocompromised mice. Metastatic penile squamous cell carcinoma (PSCC) has only a 50% response rate to first-line combination chemotherapies and there are currently no targeted-therapy approaches. Therefore, we have an urgent need in advanced-PSCC treatment to find novel therapies. Approximately half of all PSCC cases are positive for high-risk human papillomavirus (HR-HPV). Our objective was to generate HPV-positive (HPV+) and HPV-negative (HPV−) patient-derived xenograft (PDX) models and to determine the biological differences between HPV+ and HPV− disease. We generated four HPV+ and three HPV− PSCC PDX animal models by directly implanting resected patient tumor tissue into immunocompromised mice. PDX tumor tissue was found to be similar to patient tumor tissue (donor tissue) by histology and short tandem repeat fingerprinting. DNA mutations were mostly preserved in PDX tissues and similar APOBEC (apolipoprotein B mRNA editing catalytic polypeptide) mutational fractions in donor tissue and PDX tissues were noted. A higher APOBEC mutational fraction was found in HPV+ versus HPV− PDX tissues (p = 0.044), and significant transcriptomic and proteomic expression differences based on HPV status included p16 (CDKN2A), RRM2, and CDC25C. These models will allow for the direct testing of targeted therapies in PSCC and determine their response in correlation to HPV status. [ABSTRACT FROM AUTHOR]

Purpose: Metabolic reprogramming plays an important role in the tumorigenesis of clear cell renal cell carcinoma (ccRCC). Currently, positron emission tomography (PET) reporters are not used clinically to visualize altered glutamine metabolism in ccRCC, which greatly hinders detection, staging, and real-time therapeutic assessment. We sought to determine if (2S,4R)-4-[18F]fluoroglutamine ([18F]FGln) could be used to interrogate altered glutamine metabolism in ccRCC lesions in the lung. Procedures: We generated a novel ccRCC lung lesion model using the ccRCC cell line UMRC3 stably transfected with GFP and luciferase constructs. This cell line was used for characterization of [18F]FGln uptake and retention by transport analysis in cell culture and by PET/MRI (magnetic resonance imaging) in animal models. Tumor growth in animal models was monitored using bioluminescence (BLI) and MRI. After necropsy, UMRC3 tumor growth in lung tissue was verified by fluorescence imaging and histology. Results: In UMRC3 cells, [18F]FGln cell uptake was twofold higher than cell uptake in normal kidney HEK293 cells. Tracer cell uptake was reduced by 60–90% in the presence of excess glutamine in the media and by 20–50% upon treatment with V-9302, an inhibitor of the major glutamine transporter alanine-serine-cysteine transporter 2 (ASCT2). Furthermore, in UMRC3 cells, [18F]FGln cell uptake was reduced by siRNA knockdown of ASCT2 to levels obtained by the addition of excess exogenous glutamine. Conversely, [18F]FGln cellular uptake was increased in the presence of the glutaminase inhibitor CB-839. Using simultaneous PET/MRI for visualization, retention of [18F]FGln in vivo in ccRCC lung tumors was 1.5-fold greater than normal lung tissue and twofold greater than muscle. In ccRCC lung tumors, [18F]FGln retention did not change significantly upon treatment with CB-839. Conclusions: We report one of the first direct orthotopic mouse models of ccRCC lung lesions. Using PET/MR imaging, lung tumors were easily discerned from normal tissue. Higher uptake of [18F]FGln was observed in a ccRCC cell line and lung lesions compared to HEK293 cells and normal lung tissue, respectively. [18F]FGln cell uptake was modulated by exogenous glutamine, V-9302, siRNA knockdown of ASCT2, and CB-839. Interestingly, in a pilot therapeutic study with CB-839, we observed no difference in treated tumors relative to untreated controls. This was in contrast with cellular studies, where CB-839 increased glutamine uptake. [ABSTRACT FROM AUTHOR]

Immunocompromised mouse strains expressing human transgenes are being increasingly used in biomedical research. The genetic modifications in these mice cause various cellular responses, resulting in histologic features unique to each strain. The NSG-SGM3 mouse strain is similar to the commonly used NSG (NOD scid gamma) strain but expresses human transgenes encoding stem cell factor (also known as KIT ligand), granulocyte-macrophage colony-stimulating factor, and interleukin 3. This report describes 3 histopathologic features seen in these mice when they are unmanipulated or after transplantation with human CD34+ hematopoietic stem cells (HSCs), virally transduced hCD34+ HSCs, or a leukemia patient-derived xenograft. The first feature is mast cell hyperplasia: unmanipulated, naïve mice develop periductular pancreatic aggregates of murine mast cells, whereas mice given the aforementioned human cells develop a proliferative infiltrative interstitial pancreatic mast cell hyperplasia but with human mast cells. The second feature is the predisposition of NSG-SGM3 mice given these human cells to develop eosinophil hyperplasia. The third feature, secondary hemophagocytic lymphohistiocytosis/macrophage activation syndrome (HLH/MAS)–like disease, is the most pronounced in both its clinical and histopathologic presentations. As part of this disease, a small number of mice also have histiocytic infiltration of the brain and spinal cord with subsequent neurologic or vestibular signs. The presence of any of these features can confound accurate histopathologic interpretation; therefore, it is important to recognize them as strain characteristics and to differentiate them from what may be experimentally induced in the model being studied. [ABSTRACT FROM AUTHOR]

Purpose: Encorafenib + cetuximab (E+C) is an effective therapeutic option in chemorefractory BRAFV600E metastatic colorectal cancer (mCRC). However, there is a need to improve the efficacy of this molecular-targeted therapy and evaluate regimens suitable for untreated BRAFV600E in patients with mCRC., Experimental Design: We performed a series of in vivo studies using BRAFV600E mCRC tumor xenografts. Mice were randomized to receive 5-fluoruracil (5-FU), irinotecan, or oxaliplatin regimens (FOLFIRI or FOLFOX), (E+C) or the combination. Patients received long-term treatment until disease progression, with deescalation strategies used to mimic maintenance therapy. Transcriptomic changes after progression on cytotoxic chemotherapy or targeted therapy were assessed., Results: Antitumor activity of either FOLFIRI or E+C was better as first-line treatment as compared with second-line, with partial cross-resistance seen between a cytotoxic regimen and targeted therapy with an average 62% loss of efficacy for FOLFIRI after E+C and a 45% loss of efficacy of E+C after FOLFIRI (P < 0.001 for both). FOLFIRI-treated models had upregulation of epithelial-mesenchymal transition (EMT) and MAPK pathway activation, where E+C treated models had suppressed MAPK signaling. In contrast, with chemotherapy with E+C, EMT and MAPK signaling remained suppressed. FOLFOX or FOLFIRI, each in combination with E+C, were the most active first-line treatments as compared with E+C or to chemotherapy alone. Furthermore, FOLFOX in combination with E+C as first-line induction therapy, followed by E+C ± 5-FU as maintenance therapy, was the most effective strategy for long-term disease control., Conclusions: These results support the combination of cytotoxic chemotherapy and molecular-targeted therapy as a promising therapeutic approach in the first-line treatment of BRAFV600E mCRC., (©2023 The Authors; Published by the American Association for Cancer Research.)

Diverse factors contribute to the limited clinical response to radiotherapy (RT) and immunotherapy in metastatic non-small cell lung cancer (NSCLC), among which is the ability of these tumors to recruit a retinue of suppressive immune cells-such as M2 tumor-associated macrophages (TAM)-thereby establishing an immunosuppressive tumor microenvironment that contributes to tumor progression and radio resistance. M2 TAMs are activated by the STAT6 signaling pathway. Therefore, we targeted STAT6 using an antisense oligonucleotide (ASO) along with hypofractionated RT (hRT; 3 fractions of 12 Gy each) to primary tumors in three bilateral murine NSCLC models (Lewis lung carcinoma, 344SQ-parental, and anti-PD-1-resistant 344SQ lung adenocarcinomas). We found that STAT6 ASO plus hRT slowed growth of both primary and abscopal tumors, decreased lung metastases, and extended survival. Interrogating the mechanism of action showed reduced M2 macrophage tumor infiltration, enhanced TH1 polarization, improved T-cell and macrophage function, and decreased TGFβ levels. The addition of anti-PD-1 further enhanced systemic antitumor responses. These results provide a preclinical rationale for the pursuit of an alternative therapeutic approach for patients with immune-resistant NSCLC., (©2023 American Association for Cancer Research.)