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Background Cancer stem cells are thought to be responsible for the initiation and progression of cancers. In non-small cell lung cancers (NSCLCs), Hoechst 33342 dye effluxing side population (SP) cells are shown to have stem cell like properties. The oncogenic capacity of cancer stem-like cells is in part due to their ability to self-renew; however the mechanistic correlation between oncogenic pathways and self-renewal of cancer stem-like cells has remained elusive. Here we characterized the SP cells at the molecular level and evaluated its ability to generate tumors at the orthotopic site in the lung microenvironment. Further, we investigated if the self-renewal of SP cells is dependent on EGFR mediated signaling. Results SP cells were detected and isolated from multiple NSCLC cell lines (H1650, H1975, A549), as well as primary human tumor explants grown in nude mice. SP cells demonstrated stem-like properties including ability to self-renew and grow as spheres; they were able to generate primary and metastatic tumors upon orthotopic implantation into the lung of SCID mice. In vitro study revealed elevated expression of stem cell associated markers like Oct4, Sox2 and Nanog as well as demonstrated intrinsic epithelial to mesenchymal transition features in SP cells. Further, we show that abrogation of EGFR, Src and Akt signaling through pharmacological or genetic inhibitors suppresses the self-renewal growth and expansion of SP-cells and resulted in specific downregulation of Sox2 protein expression. siRNA mediated depletion of Sox2 significantly blocked the SP phenotype as well as its self-renewal capacity; whereas other transcription factors like Oct4 and Nanog played a relatively lesser role in regulating self-renewal. Interestingly, Sox2 was elevated in metastatic foci of human NSCLC samples. Conclusions Our findings suggest that Sox2 is a novel target of EGFR-Src-Akt signaling in NSCLCs that modulates self-renewal and expansion of stem-like cells from NSCLC. Therefore, the outcome of the EGFR-Src-Akt targeted therapy may rely upon the expression and function of Sox2 within the NSCLC-CSCs.

Cancer immunotherapy faces a serious challenge because of low clinical efficacy. Recently, a number of clinical studies have reported the serendipitous finding of high rates of objective clinical response when cancer vaccines are combined with chemotherapy in patients with different types of cancers. However, the mechanism of this phenomenon remains unclear. Here, we tested in mice several cancer vaccines and an adoptive T cell transfer approach to cancer immunotherapy in combination with several widely used chemotherapeutic drugs. We found that chemotherapy made tumor cells more susceptible to the cytotoxic effect of CTLs through a dramatic perforin-independent increase in permeability to GrzB released by the CTLs. This effect was mediated via upregulation of mannose-6-phosphate receptors on the surface of tumor cells and was observed in mouse and human cells. When combined with chemotherapy, CTLs raised against specific antigens were able to induce apoptosis in neighboring tumor cells that did not express those antigens. These data suggest that small numbers of CTLs could mediate a potent antitumor effect when combined with chemotherapy. In addition, these results provide a strong rationale for combining these modalities for the treatment of patients with advanced cancers.

The tumor microenvironment is composed of cellular and stromal components such as tumor cells, mesenchymal cells, immune cells, cancer associated fibroblasts and the supporting extracellular matrix. The tumor microenvironment provides crucial support for growth and progression of tumor cells and affects tumor response to therapeutic interventions. To better understand tumor biology and to develop effective cancer therapeutic agents it is important to develop preclinical platforms that can faithfully recapitulate the tumor microenvironment and the complex interaction between the tumor and its surrounding stromal elements. Drug studies performed in vitro with conventional two-dimensional cancer cell line models do not optimally represent clinical drug response as they lack true tumor heterogeneity and are often performed in static culture conditions lacking stromal tumor components that significantly influence the metabolic activity and proliferation of cells. Recent microfluidic approaches aim to overcome such obstacles with the use of cell lines derived in artificial three-dimensional supportive gels or micro-chambers. However, absence of a true tumor microenvironment and full interstitial flow, leads to less than optimal evaluation of tumor response to drug treatment. Here we report a continuous perfusion microfluidic device coupled with microscopy and image analysis for the assessment of drug effects on intact fresh tumor tissue. We have demonstrated that fine needle aspirate biopsies obtained from patient-derived xenograft models of adenocarcinoma of the lung can successfully be analyzed for their response to ex vivo drug treatment within this biopsy trapping microfluidic device, wherein a protein kinase C inhibitor, staurosporine, was used to assess tumor cell death as a proof of principle. This approach has the potential to study tumor tissue within its intact microenvironment to better understand tumor response to drug treatments and eventually to choose the most effective drug and drug combination for individual patients in a cost effective and timely manner.

Sarcomas are rare and heterogeneous mesenchymal tumors affecting both pediatric and adult populations with more than 70 recognized histologies. Doxorubicin and ifosfamide have been the main course of therapy for treatment of sarcomas; however, the response rate to these therapies is about 10-20% in metastatic setting. Toxicity with the drug combination is high, response rates remain low, and improvement in overall survival, especially in the metastatic disease, remains negligible and new agents are needed. Wee1 is a critical component of the G2/M cell cycle checkpoint control and mediates cell cycle arrest by regulating the phosphorylation of CDC2. Inhibition of Wee1 by MK1775 has been reported to enhance the cytotoxic effect of DNA damaging agents in different types of carcinomas. In this study we investigated the therapeutic efficacy of MK1775 in various sarcoma cell lines, patient-derived tumor explants ex vivo and in vivo both alone and in combination with gemcitabine, which is frequently used in the treatment of sarcomas. Our data demonstrate that MK1775 treatment as a single agent at clinically relevant concentrations leads to unscheduled entry into mitosis and initiation of apoptotic cell death in all sarcomas tested. Additionally, MK1775 significantly enhances the cytotoxic effect of gemcitabine in sarcoma cells lines with different p53 mutational status. In patient-derived bone and soft tissue sarcoma samples we showed that MK1775 alone and in combination with gemcitabine causes significant apoptotic cell death. Magnetic resonance imaging (MRI) and histopathologic studies showed that MK1775 induces significant cell death and terminal differentiation in a patient-derived xenograft mouse model of osteosarcoma in vivo. Our results together with the high safety profile of MK1775 strongly suggest that this drug can be used as a potential therapeutic agent in the treatment of both adult as well as pediatric sarcoma patients.

BackgroundMyeloid-derived suppressor cells (MDSCs) represent a diverse population of immature myeloid cells with a strong capacity to suppress the functions of NK cells and CD4+ and CD8+ T-cells in tumor immune microenvironment, thereby reducing the efficacy of immunotherapeutic drugs. MDSC depletion strategies are being investigated to determine if the immunosuppressive effects of MDSCs in the tumor microenvironment can be reduced to improve the efficacy of cancer immunotherapy. Here we employed the 3D-Explore ex vivo platform to test the efficacy of MDSC targeting agents alone and in combination with nivolumab using patient derived 3D-tumoroids which retain the tumors’ original stroma and suppressive immune landscape.Methods3D-tumoroids approximately 150 µm in size were generated from fresh patient renal cell carcinoma tumor samples, which were obtained with informed consent and relevant IRB approval. Tumoroids were treated ex vivo with a Phosphodiesterase-5 inhibitor Tadalafil, an HDAC inhibitor entinostat, a potent steroidal liver X receptor (LXR) ligand DMHCA or a selective CK2 (casein kinase II) inhibitor TBCA alone and in combination with nivolumab. Flow cytometry, high content confocal analysis and cytokine release assays were performed to monitor treatment-mediated changes in tumor immune landscape and tumor cell killing.ResultsMulti-parameter flow cytometry (14 color) analysis demonstrated the heterogeneity of tumor immune cell populations including polymorphonuclear (PMN-MDSC) and monocytic (M-MDSC) MDSCs in different patient tumor samples. Furthermore, we performed flow cytometry analysis to assess treatment-induced changes in NK and effector T-cells activation and proliferation profiles that were further correlated with drug impacts on MDSC, Tregs and myeloid cell populations in the ex vivo treatment groups. Additionally, immune cell functions were assessed by multiplex cytokine analysis and treatment-induced tumor cell killing was quantified by high content confocal imaging.ConclusionsOur data demonstrated that the ex vivo 3D-Explore is a clinically relevant platform to identify combination of immunotherapy agents capable of overcoming the unique suppressive environment developed by an individual tumor. Furthermore, the 3D-EXplore platform provides unique insight into the intact tumor immune microenvironment to develop therapeutic strategies to overcome immunosuppressive effects of MDSCs to improve the efficacy of immunotherapeutic agents in cancer.

Combinations of targeted drugs have been employed to treat sarcomas, however, response rates have not improved notably, therefore emphasizing the need for novel treatments. In addition, imaging approaches to assess therapeutic response is lacking, as currently measurable indices, such as volume and/or diameter, do not accurately correlate with changes in tumor biology. In this study, quantitative and profound analyses of magnetic resonance imaging (MRI) were developed to evaluate these as imaging biomarkers for MK1775 and Gem in an osteosarcoma xenotransplant model at early time-points following treatment. Notably, we showed that Gem and Gem+MK1775 groups had significantly inhibited tumor growth by day 4, which was presaged by elevations in mean ADC by 24 hours post treatment. Significant differences were also observed at later time points for the Gem+MK1775 combination and MK1775 therapy. ADC distribution and entropy (randomness of ADC values) were also elevated by 24 hours following therapy. Immunohistochemistry demonstrated that these treatment-related increases in ADC correlated with apoptosis and observed cell condensations (dense- and exploded bodies). These findings underline the role of ADC as a quantitative imaging biomarker for therapy-induced response and show promising clinical relevance in the sarcoma patient population.

Histone deacetylase inhibitors (HDACis) are promising anticancer drugs; however, the molecular mechanisms leading to HDACi-induced cell death have not been well understood and no clear mechanism of resistance has been elucidated to explain limited efficacy of HDACis in clinical trials. Here, we show that protein levels of checkpoint kinase 1 (Chk1), which has a major role in G(2) cell cycle checkpoint regulation, was markedly reduced at the protein and transcriptional levels in lung cancer cells treated with pan-and selective HDACis LBH589, scriptaid, valproic acid, apicidin, and MS-275. In HDACi treated cells Chk1 function was impaired as determined by decreased inhibitory phosphorylation of cdc25c and its downstream target cdc2 and increased expression of cdc25A and phosphorylated histone H3, a marker of mitotic entry. In time course experiments, Chk1 downregulation occurred after HDACi treatment, preceding apoptosis. Ectopic expression of Chk1 overcame HDACi-induced cell death, and pretreating cells with the cdc2 inhibitor purvalanol A blocked entry into mitosis and prevented cell death by HDACis. Finally, pharmacological inhibition of Chk1 showed strong synergistic effect with LBH589 in lung cancer cells. These results define a pathway through which Chk1 inhibition can mediate HDACi-induced mitotic entry and cell death and suggest that Chk1 could be an early pharmacodynamic marker to assess HDACi efficacy in clinical samples.

BackgroundCD47 is an innate immune checkpoint receptor that is overexpressed on tumor cells and contributes to immune evasion through engagement of a myeloid-lineage inhibitory protein SIRPα. Blockade of the CD47-SIRPα interaction is proved to enhance the phagocytosis of cancer cells and to induce effective antitumor immune response. Here we developed a novel ex vivo platform using fresh patient tumor samples with intact stromal components and tumor immune microenvironment to assess the therapeutic activity of immunotherapeutic drugs targeting CD47-SIRPα signaling axis in combination with the human IgG1 αPD-L1 antibody avelumab.MethodsAll tumor samples were obtained with patient consent and relevant IRB approval. Unpropagated 3D tumoroids with intact TME measuring 150 µm in size were prepared from fresh tumor samples of renal cell carcinoma using proprietary technology developed at Nilogen Oncosystems. Tumoroids prepared from each patient's tumor sample were pooled to represent the tumor heterogeneity and treated ex vivo with phrodo-labeled avelumab alone or in combination with anti-CD47 or anti-SIRPα therapeutics.ResultsMultiparameter flow analysis demonstrated tumor binding of avelumab confirming drug penetration into the intact tumor stroma that is further corroborated by high content confocal analysis. Using our confocal-based tumor cell killing assay we were able to quantify drug-induced tumor cell killing ex vivo. We further documented the impact of anti-CD47 and anti-SIRPα therapeutics on phagocytosis of dead tumor cells by tumor resident macrophages and activation of innate and adaptive effector cells by flow cytometry and confocal imaging. Additionally, pHrodo-labeled bioparticles were used to corroborate treatment-mediated changes in the phagocytic activity of tumor resident macrophages.ConclusionsIn this comprehensive study we demonstrate that the 3D-EXplore ex vivo platform can be used to assess the efficacy of therapeutic blockade of CD47/SIRPα axis on stimulation of phagocytic process within an intact tumor immune microenvironment.

BackgroundThe tumor immune microenvironment comprises a heterogeneous collection of adaptive and innate immune cells that play a critical role in immune evasion and response to immunotherapeutic agents. cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway results in activation of various immune cells promoting innate immunity in addition to senescence of cancer cells. However, the mechanisms involved in response and resistance to cGAS-STING pathway activation is not well understood. Using Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq), we explored immunological heterogeneity of tumor microenvironment in colorectal cancer and analyzed transcriptional and compositional changes of the immune landscape in response to cGAS-STING pathway activation alone and in combination with a PD-1 inhibitor nivolumab.MethodsAll human tumor samples were obtained with proper patient consent and IRB approval. Fresh patient tumor tissue was processed to generate uniform sized live 3D tumoroids measuring 150 µm in size. Treatment groups included a STING agonist, ADU-S100, alone or in combination with nivolumab. Here, we applied multi-modal CITE-seq profiling using the 10X Genomics platform to interrogate cellular responses to ex vivo treatment. Culture supernatants were collected for multiplex analysis of cytokine release in media. Additionally, flow cytometry was used to assess the activation profile of resident immune cells.ResultsMultimodal analysis of transcriptomes or proteomics at the single-cell level provided an unprecedented view of cellular diversity and enabled better understanding of how activation of STING pathway alone and in combination with nivolumab affects the TME in colorectal cancer. Flow cytometric analysis of immune cell populations isolated from 3D tumoroids demonstrated treatment mediated activation of tumor resident T-cells and changes in the innate immune cells, which coincided with marked changes in pro-and anti-inflammatory cytokine profiles determined by multiplex analysis.ConclusionsThese results demonstrate that the 3D-EXplore ex vivo tumoroid model provides a unique platform to assess the efficacy of immunotherapeutic agents and to develop novel therapeutic combinations. Furthermore, implementation of this platform in the clinical studies may also allow identifying clinically relevant biomarkers to enable the most effective treatment strategies for individual patients.

BackgroundAntibody drug conjugates (ADCs) are an effective tool for site directed delivery of cytotoxic agents to cancer cells. Tailoring of ADC-specificity to the uniqueness of a patient‘s tumor can aid in direct-targeting of tumor cells and potentially improve drug responsiveness. Here we evaluate the potential of using an ADC therapy for targeted tumor cell death and immune cell activation in combination with checkpoint inhibitors in 3D tumoroids.MethodsAll human tumor samples were obtained with proper patient consent and IRB approval. Fresh patient tumor tissue of various histologic types including CRC and NSCLC were processed to generate uniform sized live 3D tumoroids measuring 150 µm in size. Treatment groups included a conjugated ADC therapeutic antibody alone or in combination with PD-1/PD-L1 inhibitors. Culture supernatants were collected for multiplex analysis of cytokine release in media. Additionally, flow cytometry was used to assess the activation profile of resident immune cells in combination with high-content confocal imaging to determine extent of tumor cell death in the intact tumor extracellular matrix.ResultsUsing fresh patient-derived tumoroids, we observed ADC-mediated cell death and activation of immune cells within the tumor microenvironment. Production of pro-inflammatory cytokines correlated with increased activation of tumor infiltrating immune cell populations. The improved immune response led to increased tumor cell killing within the 3D tumor microenvironment observed by high-content confocal imaging.ConclusionsIn this study we demonstrate that our physiologically relevant 3D tumoroid model is an effective system to assess novel antibody drug conjugates and to develop rational drug combinations with other immuno-oncology agents. Furthermore, implementation of 3D-EX platform, in the clinical setting, may also allow for determination of the most effective combinatorial immuno-oncology treatment strategies for individualized patient care.Ethics ApprovalThe study was approved by Chesapeake IRB Pro00014313.