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Live imaging of explants containing MCFDCISO cells (H2B:mCherry, red, nuclei) with or without 578T cells (H2B:GFP, green, nuclei) showing cell motility.

Defining how interactions between tumor subpopulations contribute to invasion is essential for understanding how tumors metastasize. Here, we find that the heterogeneous expression of the transcription factor ΔNp63 confers distinct proliferative and invasive epithelial-to-mesenchymal transition (EMT) states in subpopulations that establish a leader–follower relationship to collectively invade. A ΔNp63-high EMT program coupled the ability to proliferate with an IL1α- and miR-205–dependent suppression of cellular protrusions that are required to initiate collective invasion. An alternative ΔNp63-low EMT program conferred cells with the ability to initiate and lead collective invasion. However, this ΔNp63-low EMT state triggered a collateral loss of fitness. Importantly, rare growth-suppressed ΔNp63-low EMT cells influenced tumor progression by leading the invasion of proliferative ΔNp63-high EMT cells in heterogeneous primary tumors. Thus, heterogeneous activation of distinct EMT programs promotes a mode of collective invasion that overcomes cell intrinsic phenotypic deficiencies to induce the dissemination of proliferative tumor cells.Significance:These findings reveal how an interaction between cells in different EMT states confers properties that are not induced by either EMT program alone.

Supplemental Figures S1-S5 and Supplemental Legends

Introduction: Cancer associated fibroblasts (CAFs) are a major component of the tumormicroenvironment (TME) and exhibit diverse tumorigenic functions such as immunosuppressionand extracellular matrix (ECM) remodeling. Treatment of solid tumors is often hindered by acomplex TME, which persists despite effective tumor-cell directed therapies. Therefore,treatment of solid tumors in combination with stromal-targeting therapies is essential toovercome CAF-facilitated tumor growth, and immunosuppression. Herein we interrogate theimpact of tumor stroma targeting therapeutics, in combination with nivolumab, on the efficacy oftumor cell killing (TCK) in a 3D human tumoroid ex vivo culture model (3D-EXplore). Resultingdata suggests that impairing the recruitment and activation of CAFs within the TME leads toenhanced TCK when combined with immune checkpoint blockade therapy. Materials and Methods: All patients tumor samples were collected with patient consent andrelevant IRB approval. 3D tumoroids measuring 150 microns in size were generated from freshpatient tumors including endometrial, ovarian, and colorectal cancer tissues. Tumoroids werethen treated with stromal targeting strategies for TGF-beta (galunisertib), the FGF pathway(Dovitinib), FAK inhibitors (defactinib), and cell adhesion modulators (plerixafor) alone or incombination with nivolumab for 72-hours ex vivo. Tumor responses to ex vivo treatments wereassessed using a proprietary tumor cell killing assay and 21-color flow cytometry analysis. Results and Summary: Here we evaluated the impact of stromal targeting therapeutics onCAFs and associated tumor cell killing as well as overcoming immune evasion mechanismspreserved in the tumoroid models. Furthermore, we used a multicolor flow panel to analyzewhether combination of stromal targeting drugs enhances nivolumab’s effect on the activation oftumor resident CD4, CD8 T-cells, NKT, and NK cell populations as well as on macrophagepolarization. Treatment-mediated changes in the tumor immune microenvironment was furthercorroborated by a multiplex cytokine release assay detecting GM-CSF, sCD137, IFNγ, sFas,sFasL, Granzyme A, Granzyme B, IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, MIP-1α, MIP-1β, TNF-α,Perforin in tumoroid culture media and correlated with clinicopathologic findings and PD-L1expression for individual tumnors. Further, this 3D-tumoroid platform provides unique insightinto the microenvironment of both treatment responsive and non-responsive tumors and can aidin the development of patient-centered therapeutic regimens. Citation Format: Seth Currlin, Brittney Ruedlinger, Sharon Camacho, Angie Rivera, Jasmin D'Andrea, Jared Ehrhart, Soner Altiok. 3D-EXplore platform of fresh patient tumoroids with intact TME allows assessment of the efficacy of drugs targeting the tumor stroma on ex vivo tumor immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4552.

Introduction: Recent studies showed that patients with deficient mismatch repair(dMMR)/microsatellite instability-high (MSI-H) tumors, have higher sensitivity to immune checkpointinhibitors (ICIs) compared to patients with microsatellite-stable (MSS)/microsatellite instability-low(MSI-L) tumors. However, the mechanisms of treatment responsiveness and resistance is not wellunderstood. Here, we used a novel 3D-EXpress ex vivo fresh patient tumoroid platform to assess theefficacy of nivolumab/ipilimumab combination therapy in tumors with known MSS/MSI-H status andperformed correlative studies. Materials and Methods: All tumor samples were obtained with patient consent and relevant IRBapproval. Tumoroids measuring 150 µm in size retaining tumor cell heterogeneity, tumor-residentimmune cells, stromal components, and cell-extracellular matrix interaction were prepared frompatients with endometrial and colorectal tumors among others. Tumoroid aliquots were cryopreservedin Nilogen’s tumoroid Biorepository for future studies. For the 3D-EXpress studies cryopreservedtumoroids were selected based on MSS/MSI-H status and treated ex vivo with nivolumab andipilimumab alone and in combinations for 72h. Tumor responses to treatments were evaluated by aproprietary tumor cell killing assay and changes in tumor immune microenvironment. Furthermore,tumor PD-L1 expression levels were analyzed on the associated TMA slides. Results: Treatment-induced tumor cell killing activity in intact tumoroids was assessed by a 3D high-content confocal imaging technique using a proprietary algorithm for data analysis. The impact of exvivo treatment by nivolumab and/or ipilimumab on tumor resident immune cell populations wasmonitored by a multiplex cytokine release assay analyzing release of GM-CSF, sCD137, IFNγ, sFas, sFasL,Granzyme A, Granzyme B, IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, MIP-1α, MIP-1β, TNF-α, and Perforin usingculture supernatants isolated from treated tumoroid samples. Based on the ex vivo responses tumorswere assigned to treatment sensitive and resistant groups and correlative analyses were performed withindividual tumors’ innate and adaptive immune cell populations detected by a 21-color flow cytometrypanel, in addition to tumor MSS/MSI-H and p53 status, and detailed clinicopathologic data readilyavailable for the tumors cryopreserved in the Biorepository. Conclusion: The 3D-Express platform, using cryopreserved 3D tumoroids with intact TME is an effectivetool for the assessment of rational combinations which may prove relevant in the treatment of solidtumors. Furthermore, we believe this platform is a useful tool for the pre-clinical assessment of specifictherapeutic regimens designed for individualized patient care. Citation Format: Brittney Ruedlinger, Seth Currlin, Sharon Camacho, Alliyah Humphrey, Jared Ehrhart, Soner Altiok. 3D-EXpress platform utilizing tumoroids from patients with MSS and MSI-H tumors allows rapid assessment of anti-tumor activity of immune checkpoint inhibitors and development of clinically relevant biomarkers of treatment response. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4557.

Introduction: Nilogen Oncosystems’ 3D-EXpress platform allows for the detailed characterization offreshly resected patient tumor tissue for inclusion in studies investigating rational combinations of IOtherapeutics. Incorporating data on immune cell composition, tumor cell target expression, mutationalstatus, and HPV infection within tumoroids generated from HNSCC tissues provides tremendous input toidentify candidate tissues for therapeutic testing. The 3D-EXpress platform employed here providesdetailed observations investigating tumor cell viability using confocal microscopy and immune cellactivation indicated by increases in cytokine release. Materials and Methods: 3D tumoroids measuring 150 microns in size were generated from fresh patientHNSCC resection samples collected with proper patient consent and relevant IRB approval. Tumoroidswere never enzymatically dissociated, propagated, or reassembled to maintain the intact tumormicroenvironment (TME). The resulting tumoroids were cryopreserved in the Biorepository, where eachsample had associated patient demographic data, tumor grade, stage, mutational and HPV status inaddition to a detailed tumor immune profile and a FFPE representative sample in TMA format for targetselection. Pooled tumoroids for each patient tumor were treated ex vivo both singly with a selectiveWEE1 inhibitor, AZD1775 and an ATR blocker AZD6738 alone and in combination with a PD-1 immunecheckpoint inhibitor antibody nivolumab for 72h. Results and Summary: To quantify treatment-mediated tumor cell killing we used 3D high-contentconfocal imaging and a proprietary algorithm for data analysis. Additionally, culture supernatantsisolated from treated tumoroids were used for multiplex cytokine detection to monitor changes in theTME upon ex vivo treatment. Responses to treatments were further correlated with the composition ofinnate and adaptive TIL populations within each tumor as assessed by 21-color flow cytometry inaddition to tumor mutational status, HPV infection, smoking status, as well as EGFR and PD-L1expression levels analyzed by multiplex IF on associated TMA slides. Conclusion: These results demonstrate that targeting DNA damage response and immune checkpointblockade may provide a potential combination strategy for the treatment of HNSCC. Furthermore, weshowed that our 3D-EXpress ex vivo tumoroid model provides a unique platform to rapidly assess theefficacy of drugs and drug combinations in fresh patient tumor samples with intact TME. Correlation ofex vivo drug responses with characteristics of each tumor’s immune microenvironment and detailedclinicopathological data may allow to identify clinically relevant biomarkers to enable the most effectivetreatment strategies for individual patients in the clinic. Citation Format: Jared Ehrhart, Seth Currlin, Sharon Camacho, Samantha Hoffman, Angie Rivera, Soner Altiok. 3D-EXpress ex vivo platform using a biorepository of characterized fresh patient tumoroids allows development of rational combinations with drugs targeting DNA damage response and immune checkpoint blockade. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4572.

Introduction: 3D-EXpress is a novel ex vivo drug testing platform using a Biorepository of neverdissociated, propagated, or reassembled fresh patient tumoroids with intact tumor microenvironment.Tumoroids measuring 150 µm in size retain tumor cell heterogeneity, tumor-resident innate andadaptive immune cells, stromal components, and cell-extracellular matrix interaction allowing to rapidlytest the efficacy of drugs and drug combinations targeting various components of the TME includingtumor cells, stroma, and immune cell populations. Here we employed the 3D-EXpress platform tocompare the efficacy of different drugs and drug combinations targeting KRAS and PD-1/PD-L1 immunecheckpoint ex vivo. Materials and Methods: All tumor samples were obtained with patient consent and relevant IRBapproval. Cryopreserved tumoroids with known KRAS mutation status, PD-L1 expression, detailedimmune profiles and clinicopathologic data were selected from Nilogen’s tumoroid Biorepository for theex vivo assays. 3D tumoroids were treated with vehicle only, a SOS1::KRAS inhibitor, BI 1701963, apotent, selective, and covalent KRASG12C inhibitor, MRTX849, and a KRAS G12C inhibitor, sotorasib,alone and in combination with nivolumab for 72h. Treatment-mediated changes in tumor cell killing andtumor immune microenvironment were analyzed. Results: To quantify treatment-mediated tumor cell killing activity we employed 3D high-contentconfocal imaging using a proprietary algorithm for data analysis. To monitor how ex vivo drugtreatments affect the tumor immune microenvironment culture supernatants isolated from treatedtumoroid samples were used for multiplex cytokine detection including GM-CSF, sCD137, IFNγ, sFas,sFasL, Granzyme A, Granzyme B, IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, MIP-1α, MIP-1β, TNF-α, and Perforin.We observed a significant difference in tumor responses among patient samples to ex vivo tested drugsand drug combinations, which were further correlated with tumor resident innate and adaptive immunecell populations detected by a 21-color flow cytometry panel in addition to tumor KRAS mutation status,patient smoking history, tumor characteristics as well as PD-L1 expression levels analyzed on theassociated TMA slides. Conclusion: Our data demonstrate that the 3D-Express platform, using cryopreserved 3D tumoroidswith intact TME, is an effective tool to assess the efficacy of KRAS and immune checkpoint inhibitorstargeting drugs to identify rational combination therapies and to develop clinically relevant biomarkersfor individualized patients in the future. Citation Format: Jared Ehrhart, Brittney Ruedlinger, Angie Rivera, Romanus Ezeoke, Sharon Camacho, Seth Currlin, Soner Altiok. A novel ex vivo platform, 3D-EXpress, to rapidly assess the efficacy of KRAS targeting drugs alone and in combination with nivolumab using a biorepository of fresh patient tumoroids with intact tumor microenvironment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4571.

Defining how interactions between tumor subpopulations contribute to invasion is essential for understanding how tumors metastasize. Here, we find that the heterogeneous expression of the transcription factor ΔNp63 confers distinct proliferative and invasive epithelial-to-mesenchymal transition (EMT) states in subpopulations that establish a leader–follower relationship to collectively invade. A ΔNp63-high EMT program coupled the ability to proliferate with an IL1α- and miR-205–dependent suppression of cellular protrusions that are required to initiate collective invasion. An alternative ΔNp63-low EMT program conferred cells with the ability to initiate and lead collective invasion. However, this ΔNp63-low EMT state triggered a collateral loss of fitness. Importantly, rare growth-suppressed ΔNp63-low EMT cells influenced tumor progression by leading the invasion of proliferative ΔNp63-high EMT cells in heterogeneous primary tumors. Thus, heterogeneous activation of distinct EMT programs promotes a mode of collective invasion that overcomes cell intrinsic phenotypic deficiencies to induce the dissemination of proliferative tumor cells. Significance: These findings reveal how an interaction between cells in different EMT states confers properties that are not induced by either EMT program alone.

espanolPor medio de un analisis del voto por territorio desde el ano 1953, enfocado en el sistema de partidos, el articulo examina la fortaleza o debilidad del respaldo electoral municipal y la relacion entre el apoyo obtenido, con los partidos politicos que han llegado a la presidencia; con el objetivo de hacer un examen del comportamiento de estos partidos politicos a nivel de las unidades territoriales y conocer el grado de homogeneidad territorial de los apoyos, a traves de una comparacion de los patrones de comportamiento entre los municipios, asi como en el tiempo EnglishThe article examines the strength or weakness of the municipal electoral support and the relation between the support attained and the political parties that have achieved the presidency. The article examines the vote per territory since 1953, focused on the party system with the aim of analyzing the behavior of these political parties at the territorial unit level and also with the aim of knowing the extent of territorial homogeneity of the support by means of a comparison of the behavioral patterns among municipalities and throughout time

Cells that lead collective invasion can have distinct traits and regulatory programs compared to the cells that follow them. Notably, a specific type of epithelial-to-mesenchymal transition (EMT) program we term a "trailblazer EMT" endows cells with the ability to lead collective invasion and promote the opportunistic invasion of intrinsically less invasive siblings. Here, we sought to define the regulatory programs that are responsible for inducing a trailblazer EMT in a genetically engineered mouse (GEM) model of breast cancer. Analysis of fresh tumor explants, cultured organoids and cell lines revealed that the trailblazer EMT was controlled by TGF{beta} pathway activity that induced a hybrid EMT state characterized by cells expressing E-cadherin and Vimentin. Notably, the trailblazer EMT was active in cells lacking keratin 14 expression and evidence of trailblazer EMT activation was detected in collectively invading cells in primary tumors. The trailblazer EMT program required expression of the transcription factor Fra1, which was regulated by the parallel autocrine activation of the epidermal growth factor receptor (EGFR) and extracellular signal regulated kinases (ERK) 1 and 2. Together, these results reveal that the activity of parallel TGF{beta} and EGFR pathways confers cells with the ability to lead collective invasion through the induction of a trailblazer EMT.

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells. Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.

Base excision repair (BER) is one of the most frequently used cellular DNA repair mechanisms and modulates many human pathophysiological conditions related to DNA damage. Through live cell and in vitro reconstitution experiments, we have discovered a major sub‐pathway of conventional long‐patch BER that involves formation of a 9‐nucleotide gap 5′ to the lesion. This new sub‐pathway is mediated by RECQ1 DNA helicase and ERCC1‐XPF endonuclease in cooperation with PARP1 poly(ADP‐ribose) polymerase and RPA. The novel gap formation step is employed during repair of a variety of DNA lesions, including oxidative and alkylation damage. Moreover, RECQ1 regulates PARP1 auto‐(ADP‐ribosyl)ation and the choice between long‐patch and single‐nucleotide BER, thereby modulating cellular sensitivity to DNA damage. Based on these results, we propose a revised model of long‐patch BER and a new key regulation point for pathway choice in BER. ![][1] DNA base excision repair (BER) is essential for coping with highly frequent oxidative and alkylation base damage. Identification of a novel sub‐pathway argues for a revision of mammalian long‐patch BER models and suggests a new key regulation point in BER pathway choice. [1]: /embed/graphic-1.gif

The base excision repair (BER) pathway is one of the most frequently used DNA repair mechanisms in the cell and modulates many human pathophysiological conditions related to DNA damage. Through live cell and in vitro reconstitution experiments, we have discovered a novel step in a sub-pathway of the conventional long-patch BER that involves formation of a 9 nt gap 5’ to the lesion, which is mediated by RECQ1 DNA helicase and ERCC1-XPF endonuclease in cooperation with PARP1 and RPA. The novel step is employed during repair of a variety of DNA lesions, including oxidative and alkylation damage. Moreover, RECQ1 regulates the autoribosylation status of PARP1 and the choice between long-patch and single-nucleotide BER, which regulates cellular toxicity to DNA damage. Taken together, our results propose a revised model of the long-patch BER pathway and characterize a crucial regulation point for pathway choice in BER. Citation Format: Jordan Woodrick, Sharon Camacho, Swetha Parvathaneni, Sudha Sharma, Rabindra Roy. 5’ gap-mediated long patch base excision repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-265. doi:10.1158/1538-7445.AM2017-LB-265