Immunotherapy has revolutionized cancer treatment, offering new hope for many patients. However, while some individuals show remarkable responses, the overall success rate remains limited. This has spurred interest in combination therapies, particularly with established treatments like radiation therapy (RT), to improve outcomes. RT is a cornerstone of cancer therapy and known to influence the immune landscape, yet a systematic characterization of its effects on tumor-infiltrating leukocytes (TILs) and a rationale-based therapy is still lacking. In this study, we employed a diverse set of pre-clinical syngeneic murine tumor models with varying immune profiles to investigate the immunological impact of tumor targeted RT. We observed that immunologically ‘hot’ tumors showed stronger tumor growth inhibition (TGI) after RT compared to ‘cold’ tumors. Additionally, RT induced both pro- and anti-inflammatory shifts within the tumor immune microenvironment. Importantly, RT led to an intra-tumoral increase in proliferating CD8+ T cells, while the population of proliferating macrophages was notably reduced. To identify immune-modulatory pathways that shape the response to RT across different tumor immune contexts, we tested RT in HPK1 (Hematopoietic Progenitor Kinase 1) and STING (Stimulator of Interferon Genes) deficient mice. These experiments revealed that STING deficiency compromises TGI in tumors with a high baseline population of myeloid cells expressing an interferon response signature. Moreover, we identified a synergistic effect on survival in tumor-bearing mice when combining HPK1 deficiency with RT. Thus, RT promotes expansion of cytotoxic T cells while limiting macrophage proliferation, with therapeutic outcomes strongly influenced by STING and HPK1 pathways. Collectively, these results highlight the complex interplay between RT, tumor immune microenvironment and response to therapy, offering potential avenues for novel therapeutic combinations.

G12C KP model survival data from long-term treatment with Compound A

Baseline and post-treatment PET/CT images of individual KrasLSL-G12C/wtp53fl/fl animals treated with vehicle or Compound A

A dysregulated cell cycle is a hallmark of cancer and inhibition of cyclin-dependent kinases (CDK) is a proven therapeutic strategy in treating hormone receptor-positive/HER2- breast cancer and a variety of other cancers. 18F-3'-deoxy-3'-fluorothymidine (18F-FLT) is a validated PET biomarker to measure cell proliferation. In this study, we show the utility of 18F-FLT PET imaging as a pharmcodynamic biomarker in differentiating the efficacy of PF-07104091 (CDK2-selective inhibitor) in palbociclib (CDK4/6 inhibitor)-sensitive and -resistant tumor models. 18F-FLT PET imaging was performed after 4 days of treatment with CDK inhibitors and IHC biomarkers of tumor cell proliferation (Ki67 and pRb) were evaluated for correlation. Tumor growth inhibition studies demonstrated that palbociclib was efficacious in an MCF7 model but not in an OVCAR-3 model, whereas PF-07104091 showed dose-dependent tumor growth inhibition in both MCF7 and OVCAR-3 models. Consistent with this observation, 18F-FLT PET was able to differentiate the resistance to palbociclib from sensitivity to PF-06873600 (CDK2/4/6 inhibitor) and PF-07104091 in the OVCAR-3 model. In contrast, the 18F-FLT PET biomarker showed reduced uptake in the MCF7 model after treatment with both palbociclib and PF-07104091. Similarly, PF-07104091 demonstrated reduced 18F-FLT uptake in NIBR-5493, an ovarian cancer patient-derived xenograft model. IHC biomarkers Ki67 and pRb correlated with the 18F-FLT uptake trends in all three tumor models. This work highlights the utility of 18F-FLT PET as a quantitative, noninvasive biomarker which provides whole-body information. 18F-FLT PET has potential to be a biomarker in novel CDK inhibitor clinical trials to evaluate palbociclib resistance and to identify responding and nonresponding patients.

Supplementary Methods Section

Immune checkpoint inhibitors have shown limited success in breast cancer, the most common and deadly cancer in women worldwide. Novel immune therapies, such as CD3-engaging bispecific antibodies, have shown clinical promise in hematologic malignancies. However, developing CD3 bispecifics for solid tumors has been challenging due to the difficulty in identifying tumor-specific antigens. B7-H4 is proposed as an attractive tumor-associated antigen for breast cancer therapeutics with comprehensive coverage regardless of breast cancer molecular subtype. We designed a B7-H4-targeting CD3 bispecific molecule, PF-07260437, and demonstrated B7-H4-dependent pharmacology in vitro by directing cytotoxic T-cell killing to breast cancer cell lines. Treatment of cell line- and patient-derived xenograft in vivo models of human breast cancer with PF-07260437 induced substantial tumoricidal activity, often resulting in complete responses. Mechanistically, PF-07260437 increased T-cell number and activation, leading to efficient tumor killing. Additionally, combining PF-07260437 with standard of care (palbociclib plus fulvestrant) and a checkpoint inhibitor (anti-PD-1) showed combinatorial benefits in an immune-competent in vivo model. Clinically relevant noninvasive PET/CT imaging with a CD8-targeting tracer demonstrated PF-07260437-mediated increases in intratumoral CD8 T cells, highlighting the utility of CD8-PET technology to potentially assess biomarker changes in the clinic. Finally, the manageable toxicity profile of PF-07260437 was highlighted in an exploratory toxicology study in cynomolgus monkeys. These data support the clinical testing of PF-07260437 for treating B7-H4-expressing solid tumors, including breast cancer.

Comparative activity data for Compound A and sotorasib

Summarized G12C KP survival data from long-term treatment with Compound A

We present a rigorous validation strategy to evaluate the performance of Ultivue multiplex immunofluorescence panels. We have quantified the accuracy and precision of four different multiplex panels (three human and one mouse) in tumor specimens with varying levels of T cell density. Our results show that Ultivue panels are typically accurate wherein the relative difference in cell proportion between a multiplex image and a 1-plex image is less than 20% for a given biomarker. Ultivue panels exhibited relatively high intra-run precision (CV ≤ 25%) and relatively low inter-run precision (CV >> 25%) which can be remedied by using local intensity thresholding to gate biomarker positivity. We also evaluated the reproducibility of cell–cell distance estimates measured from multiplex images which show high intra- and inter-run precision. We introduce a new metric, multiplex labeling efficiency, which can be used to benchmark the overall fidelity of the multiplex data across multiple batch runs. Taken together our results provide a comprehensive characterization of Ultivue panels and offer practical guidelines for analyzing multiplex images.

MUM images of trogocytic tubules.The movie corresponds to Figure 4B. Images of trastuzumab (left panels) or trastuzumab and MEM-GFP (red and green, respectively; right panels) from the cameras set at focal planes of ~ 1.2 µm and 1.8 µm in the MUM configuration are displayed. The cyan line in the left panels indicates the approximate edge of the macrophage, obtained by averaging and thresholding the MEM-GFP images. The movie pauses for 2 seconds at different timepoints to indicate the same features of the tubulation process (yellow arrows) presented as individual frames in Figure 4B. Time on the upper left is shown in minutes:seconds format. The movie plays at a speed of 27x real-time. Scale bar = 5 µm.

Live cell imaging of macrophage-mediated trogocytosis. The movie shows trastuzumab from live, opsonized MDA-MB-453 cells being trogocytosed by CD45-labeled J774A.1 macrophages. Upper left panel shows signal from Alexa 555-labeled trastuzumab, upper right panel shows CD45 signal, lower left panel shows the transmitted light image and the lower right image shows the overlay of the upper two panels. The sample was imaged in the presence of caspase 3/7 detection reagent for which the signal is also detected in the CD45 channel. The movie plays at a speed of 340x real-time. Scale bar = 5 µm.

Transmitted-light imaging of the fate of an SK-BR-3 cell in a J774A.1:SK-BR-3 co-culture in the presence of trastuzumab. The left panel in the movie tracks an SK-BR-3 cell (yellow box) amongst J774A.1 macrophages in the presence of 1 µg/ml trastuzumab over 3 days. The right panel traces the absolute displacement of the tracked SK-BR-3 cell in the flask over time. The title on the right panel shows the time (days) and the speed at which the movie is being played (2700x real-time until 00:42 and 900x real-time after 00:42).

Two-color live cell imaging of a J774A.1 macrophage phagocytosing MDA-MB-453 cancer cells. Macrophages with surface-labeled CD45 (green) engulf three trastuzumab-opsonized MDA-MB-453 cells (red). Time is indicated at the top left in minutes:seconds format. The movie plays at a speed of 340x real-time

Transmitted-light imaging of the fate of an SK-BR-3 cell in a RAW264.7:SK-BR-3 co-culture in the presence of trastuzumab. Movie shows the fate of an SK-BR-3 cell as in Movie S2, but with RAW264.7 macrophages as the effector cells. The title on the right panel shows the time in days - hours:minutes format. The movie plays at a speed of 1400x real-time.

Supplemental Figure S1. Concentration versus time profiles of ipilimumab and nivolumab following four weekly intravenous dose administrations of 15 mg/kg or 20 mg/kg, respectively. Supplementary Figure S2. Hematoxylin and eosin stained sections of tissues from control and monkeys dosed with ipilimumab and nivolumab. Supplementary Figure S3. Quantification of mononuclear cell infiltrates from IHC images of heart sections.

Abstract KRAS is one of the most commonly mutated oncogenes in lung, colorectal, and pancreatic cancers. Recent clinical trials directly targeting KRAS G12C presented encouraging results for a large population of non–small cell lung cancer (NSCLC), but resistance to treatment is a concern. Continued exploration of new inhibitors and preclinical models is needed to address resistance mechanisms and improve duration of patient responses. To further enable the development of KRAS G12C inhibitors, we present a preclinical framework involving translational, non-invasive imaging modalities (CT and PET) and histopathology in a conventional xenograft model and a novel KRAS G12C knock-in mouse model of NSCLC. We utilized an in-house developed KRAS G12C inhibitor (Compound A) as a tool to demonstrate the value of this framework in studying in vivo pharmacokinetic/pharmacodynamic (PK/PD) relationship and anti-tumor efficacy. We characterized the Kras G12C-driven genetically engineered mouse model (GEMM) and identify tumor growth and signaling differences compared to its Kras G12D-driven counterpart. We also find that Compound A has comparable efficacy to sotorasib in the Kras G12C-driven lung tumors arising in the GEMM, but like observations in the clinic, some tumors inevitably progress on treatment. These findings establish a foundation for evaluating future KRAS G12C inhibitors that is not limited to xenograft studies and can be applied in a translationally relevant mouse model that mirrors human disease progression and resistance.

Transmitted-light imaging of an SK-BR-3 cell in a human macrophage co-culture in the presence of trastuzumab. Movie shows the fate of an SK-BR-3 cell as in Movie S2, but with human macrophages as effector cells. Purified human monocytes were plated six days prior to the addition of cancer cells. The title on the panel indicates the time progression in days - hours:minutes format. The movie plays at a speed of 3600x real-time.

Table S1. Mean ({plus minus}SD) Toxicokinetic Parameters for Ipilimumab and Novolumab in Cynomolgus Monkeys Table S2 - Differentially expressed genes in the heart of ipilimumab and nivolumab treated cynos (adjusted P-value {less than or equal to} 0.05) Table S3 - Top 50 enriched pathways identified by IPA when using the data set of 882 analysis ready differentially expressed genes (adjusted P-value {less than or equal to} 0.05). Ratio: number of DE genes in a pathway divided by the number of genes comprised in the same pathway. Table S4: Top 25 networks identified based upon differentially expressed genes (adjusted P-value {less than or equal to} 0.05) in ipilimumab and nivolumab treated cynos. Table S5: Regulator effects networks generated using genes that are differentially expressed (adjusted P-value {less than or equal to} 0.05) in ipilimumab and nivolumab treated cynos. Upstream regulator and diseae& function cutoffs were each log P-value {less than or equal to} 10 and a z-score {greater than or equal to} |3| Table S6 - Differential expression of myocarditis- associated genes in the heart of ipilimumab and nivolumab treated cynos