Syntrix Biosystems (United States)

Engineered heart tissues (EHTs) formed around flexible pillars are used to measure the contraction force of myocytes. When based on cardiac cells derived from human induced pluripotent stem cells (hiPSCs), EHTs capture human cardiac physiology and drug responses in vitro. However, variability in contractile function often arises due to variation in tissue positioning on the pillar. Here, novel tapered pillars are introduced to achieve spatial confinement of tissues in EHT devices. The devices are fabricated by moulding polydimethylsiloxane (PDMS) into micromachined tapered cavities of a silicon substrate. The symmetrically‐tapered geometry, with the minimum cross‐section at the pillar mid‐height, restricts tissue movement outside of the indented area. This increases sensitivity and accuracy of tissue contractile readout, providing high reproducibility with reduced variability between data points. Design and stiffness of tapered pillars are investigated to determine the optimal mechanical environment, obtain accurate contractile measurements, and achieve long‐term culture of EHTs. Results show that tapered pillars provide superior confinement efficiency (over 90%) compared to straight pillars (30%), with tissue confinement directly correlated to pillar geometry rather than stiffness. The optimized precision in force readouts and long‐term tissue studies enables higher sensitivity in the detection of contractile responses to drugs or diseases.

Inhibitors of cyclin-dependent kinase 4 and 6 (CDK4/6) are approved for the treatment of locally advanced or metastatic breast cancer, but not for melanoma. In this study, we evaluated the effectiveness of the CDK4/6 inhibitor, palbociclib, the CDK2 inhibitor, PF-07104091, the dual CXCR1 and CXCR2 (CXCR1/2) antagonist, SX-682, and the combination of these inhibitors for effective treatment of melanoma in preclinical models. Both palbociclib and SX-682 inhibited the growth of BRAFWT/NRASWT B16-F10 and NRASmut 1014 melanoma tumors and in both models, SX-682 created a more anti-tumor immune microenvironment. The combination effect was additive in the B16F10 model, but not in the 1014 model. In the B16F10 model, the addition of the CDK2 inhibitor, PF-07104091, overcame B16F10 acquired resistance to CDK4/6 inhibitors by suppressing the induction of cyclin D1 and E1 expression by palbociclib. In the less responsive 1014 cells, cyclin D1 was reduced, but cyclin E1 was induced in response to PF-07104091. However, in both models, combined treatment with palbociclib and PF-07104091 markedly suppressed cyclin A2, cyclin D1, cyclin E1 and pRB-S807/S811. Combining CDK4/6 and CDK2 inhibitors with the CXCR1/2 antagonist, SX-682, halted B16F10 tumor growth by blocking tumor cell proliferation and increasing the anti-tumor immune response in the tumor microenvironment. The combination of all three inhibitors resulted in a tumor microenvironment characterized by increased IFNγ-producing CD4+ T cells, decreased CD4+FOXP3+ T regulatory cells (Tregs), and decreased IL-10-producing CD4+ T cells. This combination also decreased the percentage of CD8+ T cells that expressed PD-1 or TIM-3 and increased the ratio of MHCII+F4/80+ M1-like macrophages to CD206+F4/80+ M2-like macrophages. These data suggest that inhibiting CDK4/6 and CDK2, combined with antagonism of CXCR1/2, may be an effective treatment for BRAF wild-type melanoma tumors and NRAS mutant melanoma tumors that express Rb and are resistant to immune checkpoint inhibitors.

ABSTRACTThe noninflamed microenvironment in prostate cancer represents a barrier to immunotherapy. Genetic alterations underlying cancer cell-intrinsic oncogenic signaling have emerging roles in shaping the immune landscape. Recently, we identified Pygopus 2 (PYGO2) as the driver oncogene for the amplicon at 1q21.3. Here, using transgenic mouse models of metastatic prostate adenocarcinoma, we found that Pygo2 deletion decelerated tumor progression, diminished metastases, and extended survival. Pygo2 loss augmented the infiltration of cytotoxic T lymphocytes (CTLs) and sensitized tumor cells to T cell killing. Mechanistically, Pygo2 orchestrated a p53/Sp1/Kit/Ido1 signaling network to foster a microenvironment hostile to CTLs. Genetic or pharmacological inhibition of Pygo2 enhanced the anti-tumor efficacy of immunotherapies using immune checkpoint blockade, adoptive cell transfer, or myeloid-derived suppressor cell inhibitors. In human prostate cancer samples, Pygo2 expression was inversely correlated with CD8+ T cells. Our results highlight a promising path to improving immunotherapy with targeted therapy for lethal prostate cancer.

TPS3638 Background: Refractory RAS mutated MSS mCRC represents a critical unmet need with minimal response to immune checkpoint blockade (ICB). Preclinical CRC models reveal that KRAS mutations activate the CXCR2 axis promoting an immunosuppressive tumor microenvironment (TME). This occurs via KRAS repression of interferon regulatory factor 2 (IRF2) resulting in upregulation of CXCL3 chemokines, which bind CXCR2 and recruit myeloid-derived suppressor cells (MDSC; Liao et al, Cancer Cell 2019; Grover et al, Cancer Discovery, 2022). SX-682 is a novel oral small-molecule inhibitor of the CXCR1/2 chemokine receptors involved in MDSC-recruitment to the TME. This proof-of-concept study investigates whether de novo immunotherapy resistance in MSS RAS mutated mCRC can be overcome by treatment with a small molecule CXCR1/2 antagonist in combination with anti-PD-1 therapy. Methods: STOPTRAFFIC-1 (NCT04599140, enrolling) is a phase I/II, open-label, dose-escalation/dose expansion study of SX-682 in combination with nivolumab to evaluate safety and clinical activity for patients (pts) with refractory RAS ( KRAS and NRAS) mutated MSS mCRC. Key eligibility criteria: patients (pts) with MSS mCRC with measurable disease, progression or intolerance to at least 2 prior lines of standard therapy, and ECOG 0 or 1. Pts will receive SX-682 (5 dose levels: 25 mg, 50 mg, 100mg, 200mg, or 400 mg by mouth twice daily) administered in an 8-week cycle with intravenous nivolumab (480 mg) on days 1 and 29 of each cycle. Adverse events are assessed according to CTCAE v5.0. Response assessments (per RECIST) occur every 8 weeks. In dose escalation, pts enter a 3-week monotherapy safety run-in of SX-682 followed by 3-week combination with nivolumab for a six-week dose-limiting toxicity (DLT) period. Cohorts 1-4 have been completed without DLT. Enrollment to cohort 5 began in January 2022. Following determination of maximum tolerated dose (MTD), dose expansion design is a Simon's optimal two-stage design. Efficacy will be assessed in the first 15 pts with a requirement of at least 2 responses in order to enroll 14 additional pts (N=29) in the second stage. Pre- and on-treatment tissue biopsies will be collected in the expansion phase. The primary objectives are to determine the safety profile of SX-682 alone and in combination with nivolumab, including the MTD, recommended phase 2 dose, and the DLT. The secondary objectives include overall response rate, progression-free survival, overall survival, and pharmacokinetic profiles of SX-682. Translational analyses include correlations of clinical outcomes with genomic and immune biomarkers from paired tissue and plasma samples. Clinical trial information: NCT04599140.

TPS631 Background: Survival outcomes for advanced pancreatic ductal adenocarcinoma (PDAC) remain dismal despite improvements in systemic therapy regimens developed over the past decade. In addition, current first-line therapies result in cumulative cytopenias and neuropathy, highlighting the need for more effective, less toxic maintenance treatment strategies. There are currently no standard approved maintenance treatments for patients with PDAC not associated with BRCA or DNA-repair mutations. Pre-clinical data suggest a potential synergistic effect of combining blockade of CXC chemokine receptors (CXCR) with immunotherapy or chemotherapy in pancreatic cancer1,2. We are currently conducting a Phase I study (NCT04477343) evaluating SX-682, an oral CXCR1/2 inhibitor, and Nivolumab as maintenance treatment for advanced PDAC. Methods: This is an open-label, dose escalation Phase I clinical trial evaluating the combination of SX-682 and Nivolumab. Patients must have histologically confirmed unresectable PDAC and have completed at least 16 weeks of first-line chemotherapy with disease stability or treatment response at time of enrollment. Radiographically measurable disease must be present per iRECIST criteria. Patients receive a 3-week run-in phase of twice-daily dosing of SX-682, followed by combination of twice-daily dosed SX-682 and every 2-week Nivolumab (240 mg IV). Dose finding of SX-682 is performed using Bayesian optimal interval (BOIN) design to determine the maximum tolerated dose (MTD) when combined with Nivolumab. Pre-treatment and one on treatment (Day 28-35) biopsies are required for enrollment to evaluate change in tumor microenvironment immune cell composition by single cell-RNA sequencing, flow cytometry, RNA RT-qPCR, and IHC. The primary endpoint is to determine MTD; the key secondary endpoint is progression-free survival (PFS), defined as the time from enrollment to progression via iRECIST criteria or death. Nine of a planned 20 patients have been enrolled. Dose-level 1 (SX-682 50 mg BID) completed enrollment without dose-limiting toxicity (DLT). Dose-level 2, which commenced in June 2021, (SX-682 100 mg BID) is without DLTs, but has not completed enrollment at time of abstract submission. Nywening TM, Belt BA, Cullinan DR, et al. Targeting both tumour-associated CXCR2(+) neutrophils and CCR2(+) macrophages disrupts myeloid recruitment and improves chemotherapeutic responses in pancreatic ductal adenocarcinoma. 1) Gut. 2018;67(6):1112-1123. Steele CW, Karim SA, Leach JDG, et al. CXCR2 Inhibition Profoundly Suppresses Metastases and Augments Immunotherapy in Pancreatic Ductal Adenocarcinoma. 2) Cancer Cell. 2016;29(6):832-845. Clinical trial information: NCT04477343.

There is an unmet clinical need for stratification of breast lesions as indolent or aggressive to tailor treatment. Here, single-cell transcriptomics and multiparametric imaging applied to a mouse model of breast cancer reveals that the aggressive tumor niche is characterized by an expanded basal-like population, specialization of tumor subpopulations, and mixed-lineage tumor cells potentially serving as a transition state between luminal and basal phenotypes. Despite vast tumor cell-intrinsic differences, aggressive and indolent tumor cells are functionally indistinguishable once isolated from their local niche, suggesting a role for non-tumor collaborators in determining aggressiveness. Aggressive lesions harbor fewer total but more suppressed-like T cells, and elevated tumor-promoting neutrophils and IL-17 signaling, disruption of which increase tumor latency and reduce the number of aggressive lesions. Our study provides insight into tumor-immune features distinguishing indolent from aggressive lesions, identifies heterogeneous populations comprising these lesions, and supports a role for IL-17 signaling in aggressive progression.

Simple SummaryInnovative strategies to reduce immune suppression and activate tumor-specific immunity are needed to help patients who do not respond or become resistant to immune checkpoint blockade therapies. In this study, we demonstrate that the addition of a cancer vaccine targeting a tumor-associated antigen to a checkpoint inhibitor-based immunotherapy induces greater numbers of proliferative, activated, and cytotoxic tumor-infiltrating T cells, leading to improved antitumor activity in tumors otherwise resistant to immunotherapy. Our results provide the rationale for the addition of cancer vaccines in combination immunotherapy approaches being evaluated in the clinic.Resistance to immune checkpoint blockade therapy has spurred the development of novel combinations of drugs tailored to specific cancer types, including non-inflamed tumors with low T-cell infiltration. Cancer vaccines can potentially be utilized as part of these combination immunotherapies to enhance antitumor efficacy through the expansion of tumor-reactive T cells. Utilizing murine models of colon and mammary carcinoma, here we investigated the effect of adding a recombinant adenovirus-based vaccine targeting tumor-associated antigens with an IL-15 super agonist adjuvant to a multimodal regimen consisting of a bifunctional anti-PD-L1/TGF-βRII agent along with a CXCR1/2 inhibitor. We demonstrate that the addition of vaccine induced a greater tumor infiltration with T cells highly positive for markers of proliferation and cytotoxicity. In addition to this enhancement of cytotoxic T cells, combination therapy showed a restructured tumor microenvironment with reduced Tregs and CD11b+Ly6G+ myeloid cells. Tumor-infiltrating immune cells exhibited an upregulation of gene signatures characteristic of a Th1 response and presented with a more diverse T-cell receptor (TCR) repertoire. These results provide the rationale for the addition of vaccine-to-immune checkpoint blockade-based therapies being tested in the clinic.

Abstract There is an unmet clinical need for stratification of breast lesions as indolent or aggressive to tailor treatment. Here, single-cell transcriptomics and multiparametric imaging applied to a mouse model of breast cancer revealed that the aggressive tumor niche is characterized by an expanded basal-like population, specialization of tumor subpopulations, and mixed-lineage tumor cells potentially serving as a transition state between luminal and basal phenotypes. Despite vast tumor cell-intrinsic differences, aggressive and indolent tumor cells were functionally indistinguishable once isolated from their local niche, suggesting a role for non-tumor collaborators in determining aggressiveness. Aggressive lesions harbored fewer total but more suppressed-like T cells, and elevated tumor-promoting neutrophils and IL-17 signaling, disruption of which increased tumor latency and reduced the number of aggressive lesions. Our study provides insight into tumor-immune features distinguishing indolent from aggressive lesions, identifies heterogeneous populations comprising these lesions, and supports a role for IL-17 signaling in aggressive progression.