Background: We have an incomplete understanding of the differences between cancer stem cells (CSCs) in human papillomavirus-positive (HPV-positive) and -negative (HPV-negative) head and neck squamous cell cancer (HNSCC). The PI3K pathway has the most frequent activating genetic events in HNSCC (especially HPV-positive driven), but the differential signaling between CSCs and non-CSCs is also unknown. Methods: We addressed these unresolved questions using CSCs identified from 10 HNSCC patient-derived xenografts (PDXs). Sored populations were serially passaged in nude mice to evaluate tumorigenicity and tumor recapitulation. The transcription profile of HNSCC CSCs was characterized by mRNA sequencing, and the susceptibility of CSCs to therapy was investigated using an in vivo model. SOX2 transcriptional activity was used to follow the asymmetric division of PDX-derived CSCs. All statistical tests were two-sided. Results: CSCs were enriched by high aldehyde dehydrogenase (ALDH) activity and CD44 expression and were similar between HPV-positive and HPV-negative cases (percent tumor formation injecting 1x10 3 cells: ALDH þ CD44 high ¼ 65.8%,
Wnt pathway activation maintains the cancer stem cell (CSC) phenotype and promotes tumor progression, making it an attractive target for anti‐cancer therapy. Wnt signaling at the tumor and tumor microenvironment (TME) front have not been investigated in depth in head and neck squamous cell carcinoma (HNSCC). In a cohort of 48 HNSCCs, increased Wnt signaling, including Wnt genes (AXIN2, LGR6, WISP1) and stem cell factors (RET, SOX5, KIT), were associated with a more advanced clinical stage. Key Wnt pathway proteins were most abundant at the cancer epithelial‐stromal boundary. To investigate these observations, we generated three pairs of cancer‐cancer associated fibroblast (CAF) cell lines derived from the same HNSCC patients. 3D co‐culture of cancer spheres and CAFs mimicked these in vivo interactions, and using these we observed increased expression of Wnt genes (eg, WNT3A, WNT7A, WNT16) in both compartments. Of these Wnt ligands, we found Wnt3a, and less consistently Wnt16, activated Wnt signaling in both cancer cells and CAFs. Wnt activation increased CSC characteristics like sphere formation and invasiveness, which was further regulated by the presence of CAFs. Time lapse microscopy also revealed preferential Wnt activation of cancer cells. Wnt inhibitors, OMP‐18R5 and OMP‐54F28, significantly reduced growth of HNSCC patient‐derived xenografts and suppressed Wnt activation at the tumor epithelial‐stromal boundary. Taken together, our findings suggest that Wnt signaling is initiated in cancer cells which then activate CAFs, and in turn perpetuate a paracrine signaling loop. This suggests that targeting Wnt signaling in the TME is essential.
In this report, we describe in detail the evolving procedures to optimize humanized mouse cohort generation, including optimal conditioning, choice of lineage for engraftment, threshold for successful engraftment, HNSCC tumor implantation, and immune and stroma cell analyses. We developed a dual infusion protocol of human hematopoietic stem and progenitor cells (HSPCs) and mesenchymal stem cells (MSCs), leading to incremental human bone marrow engraftment, and exponential increase in mature peripheral human immune cells, and intratumor homing that includes a more complete lineage reconstitution. Additionally, we have identified practical rules to predict successful HSPC/MSC expansion, and a peripheral human cell threshold associated with bone marrow engraftment, both of which will optimize cohort generation and management. The tremendous advances in immune therapy in cancer have made the need for appropriate and standardized models more acute than ever, and therefore, we anticipate that this manuscript will have an immediate impact in cancer-related research. The need for more representative tools to investigate the human tumor microenvironment (TME) has led to the development of humanized mouse models. However, the difficulty of immune system engraftment and minimal human immune cell infiltration into implanted xenografts are major challenges. We have developed an improved method for generating mismatched humanized mice (mHM), using a dual infusion of human HSPCs and MSCs, isolated from cord blood and expanded in vitro. Engraftment with both HSPCs and MSCs produces mice with almost twice the percentage of human immune cells in their bone marrow, compared to mice engrafted with HSPCs alone, and yields 9- to 38-fold higher levels of mature peripheral human immune cells. We identified a peripheral mHM blood human B cell threshold that predicts an optimal degree of mouse bone marrow humanization. When head and neck squamous cell carcinoma (HNSCC) tumors are implanted on the flanks of HSPC-MSC engrafted mice, human T cells, B cells, and macrophages infiltrate the stroma of these tumors at 2- to 8-fold higher ratios. In dually HSPC-MSC engrafted mice we also more frequently observed additional types of immune cells, including regulatory T cells, cytotoxic T cells, and MDSCs. Higher humanization was associated with in vivo response to immune-directed therapy. The complex immune environment arising in tumors from dually HSPC-MSC engrafted mice better resembles that of the originating patient's tumor, suggesting an enhanced capability to accurately recapitulate a human TME.
Salivary gland cancers (SGC) frequently present with distant metastases many years after diagnosis, suggesting a cancer stem cell (CSC) subpopulation that initiates late recurrences; however, current models are limited both in their availability and suitability to characterize these rare cells. Patient-derived xenografts (PDX) were generated by engrafting patient tissue onto nude mice from one acinic cell carcinoma (AciCC), four adenoid cystic carcinoma (ACC), and three mucoepidermoid carcinoma (MEC) cases, which were derived from successive relapses from the same MEC patient. Patient and PDX samples were analyzed by RNA-seq and Exome-seq. Sphere formation potential and tumorigenicity was assessed by sorting for Aldefluor (ALDH) activity and CD44-expressing subpopulations. For successive MEC relapses we found a time-dependent increase in CSCs (ALDHCD44), increasing from 0.2% to 4.5% (=0.033), but more importantly we observed an increase in individual CSC sphere formation and tumorigenic potential. A 50% increase in mutational burden was documented in subsequent MEC tumors, and this was associated with increased expression of tumor-promoting genes (, and ), decreased expression of tumor-suppressor genes (, and ), and higher expression of CSC-related proteins such as SOX2, MYC, and ALDH1A1. Finally, genomic analyses identified a novel- fusion in an ACC tumor and confirmed previously reported fusions (- and - Sequential MEC PDX models preserved key patient features and enabled the identification of genetic events putatively contributing to increases in both CSC proportion and intrinsic tumorigenicity, which mirrored the patient's clinical course. .
Cancer stem cells (CSC) drive growth, therapy resistance, and recurrence in head and neck squamous cell carcinoma (HNSCC). Regulation of protein translation is crucial for normal stem cells and CSCs; its inhibition could disrupt stemness properties, but translation inhibitors are limited clinically due to toxicity. SVC112 is a synthetic derivative of bouvardin, a plant-derived translation elongation inhibitor. SVC112 had greater antiproliferative effects on HNSCC cells compared with the FDAapproved translation inhibitor omacetaxine mepesuccinate (HHT). SVC112 preferentially inhibited cancer cells compared with patient-matched cancer-associated fibroblasts, whereas HHT was equally toxic to both. SVC112 reduced sphere formation by cell lines and CSCs. SVC112 alone inhibited the growth of patient-derived xenografts (PDX), and SVC112 combined with radiation resulted in tumor regression in HPV-positive and HPV-negative HNSCC PDXs. Notably, CSC depletion after SVC112 correlated with tumor response. SVC112 preferentially impeded ribosomal processing of mRNAs critical for stress response and decreased CSC-related proteins including Myc and Sox2. SVC112 increased cell-cycle progression delay and slowed DNA repair following radiation, enhancing colony and sphere formation radiation effects. In summary, these data demonstrate that SVC112 suppresses CSC-related proteins, enhances the effects of radiation, and blocks growth of HNSCC PDXs by inhibiting CSCs.Significance: Inhibiting protein elongation with SVC112 reduces tumor growth in head and neck squamous cell carcinoma and increases the effects of radiation by targeting the cancer stem cell pool.
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