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Age is among the main risk factors for cancer and any cancer study in adults is faced with an aging tissue and organism. Yet, pre-clinical studies are carried out using young mice and are not able address the impact of aging and associated comorbidities on disease biology and treatment outcomes. Here, we discuss the limitations of current mouse cancer models and suggest strategies for developing novel models to address these major gaps in knowledge and experimental approaches.

Background The expansion of hematopoietic stem cells carrying recurrent somatic mutations, termed clonal hematopoiesis (CH), is common in elderly individuals and is associated with increased risk of myeloid malignancy and all-cause mortality. Though chemotherapy is a known risk factor for developing CH, how myelosuppressive therapies affect the short-term dynamics of CH remains incompletely understood. Most studies have been limited by retrospective design, heterogeneous patient populations, varied techniques to identifying CH, and analysis of single timepoints. Methods We examined serial samples from 40 older women with triple-negative or hormone receptor–positive breast cancer treated on the prospective ADjuVANt Chemotherapy in the Elderly trial to evaluate the prevalence and dynamics of CH at baseline and throughout chemotherapy (6 and 12 weeks). Results CH was detected in 44% of patients at baseline and in 53% at any timepoint. Baseline patient characteristics were not associated with CH. Over the course of treatment, mutations exhibited a variety of dynamics, including emergence, expansion, contraction, and disappearance. All mutations in TP53 (n = 3) and PPM1D (n = 4), genes that regulate the DNA damage response, either became detectable or expanded over the course of treatment. Neutropenia was more common in patients with CH, particularly when the mutations became detectable during treatment, and CH was significantly associated with cyclophosphamide dose reductions and holds (P = .02). Conclusions Our study shows that CH is common, dynamic, and of potential clinical significance in this population. Our results should stimulate larger efforts to understand the biological and clinical importance of CH in solid tumor malignancies. Trial Registration ClinicalTrials.gov (https://clinicaltrials.gov/ct2/show/NCT03858322). Clinical trial registration number: NCT03858322.

Supplementary Figure 4 shows hierarchical clustering of the most differentially expressed genes and GSEA graphs showing enriched pathways associated with age and treatment for figure 4

PDF file - 90K, EGF and IGF-1 Together Modulate Viability and Expression Profile of Responder Cells.

Various Annotations of ER+ Breast Cancer Cell Lines.

PDF file 401K, List of p38MAPK-dependent SASP factors and their overlap with publically available expression analyses from breast cancer-associated microenvironments

PDF file - 287K, Characterization of responding tumors in LBC and TNBC systemic environments suggests different mechanisms of instigation

Ras Pathway Activation upon Rescue with wild-type or GAP-mutant DAB2IP and RASAL2.

Supplementary Figure 3 shows the gating strategy and absolute numbers of CD8 cell subsets in the blood, spleen, and tumor to generate the heatmaps in figure 3

PDF file - 279K, EGFR/IGFR Inhibitors do not Affect the Phenotype of Responding Tumor Cells in Animals Bearing Control Matrigel.

PDF file 62K, legends for supplemental figures

Supplementary Table 1 provides a list of the antibodies used in this study

Supplementary Figure 5 shows a heatmap of the most differentially expressed genes in TNBC under 40 and over 65 using the METABRIC dataset for figure 5

PDF file - 117K, Tumor specimen from a patient with with clear cell renal cell carcinoma responders to the LBC environment in the systemic instigation xenograft model

PDF file - 159K, Identification of Instigating Human Breast Tumor Surgical Specimens.

PDF file - 319K, Tumor specimens from women with luminal breast cancer used to identify instigators, non-instigators, or responders in the systemic instigation xenograft model

PDF file - 508K, Tumor Microenvironment of Hosts with TNBC Expresses EGF and IGF-1 and Supports Responding Tumor Cell Malignancy.

PDF file 229K, qRT-PCR validation of RNA-seq analysis and TNFalpha ELISA on p38i treated mice

PDF file - 220K, Response to the Pro-Tumorigenic TNBC Environment is Not Oncotype Specific.

PDF file - 181K, Instigating Tumors Facilitate Responder Growth in the Absence of "Self-seeding".

Supplementary Figure 2 shows PD-L1 and MHCI upregulation after IFNg stimulation in the 4T1 and Met1 cells lines, experimental schematic, tumor volume at the start of treatment, and graphs of individual tumor growth for figure 2

Supplementary Figure Legends

PDF file - 90K

PDF file - 166K, Efficiency of BMC transplantation and tissue recruitment of BMCs; effects of LBC environment on responding tumor cell CD24 enrichment and circulating platelet counts

PDF file 545K, SASP upregulation following bleomycin, NaB or replication-induced senescence. SASP mRNA stabilization in NaB-treated BJs and in bleomycin-treated IMR90s

Supplementary Figure 6 shows schematics for experimental set-up, tumor volume at the start of treatment, and graphs showing individual tumor growth for figure 6

Supplementary File 1 lists the most differentially expressed genes from the RNAseq analysis

PDF file - 89K, Aspirin treatment does not affect growth kinetics of the LBC instigator or circulating platelet counts

Immune checkpoint blockade (ICB) therapy, which targets T cell–inhibitory receptors, has revolutionized cancer treatment. Among the breast cancer subtypes, evaluation of ICB has been of greatest interest in triple-negative breast cancer (TNBC) due to its immunogenicity, as evidenced by the presence of tumor-infiltrating lymphocytes and elevated PD-L1 expression relative to other subtypes. TNBC incidence is equally distributed across the age spectrum, affecting 10% to 15% of women in all age groups. Here we report that increased immune dysfunction with age limits ICB efficacy in aged TNBC-bearing mice. The tumor microenvironment in both aged mice and patients with TNBC shows decreased IFN signaling and antigen presentation, suggesting failed innate immune activation with age. Triggering innate immune priming with a STING agonist restored response to ICB in aged mice. Our data implicate age-related immune dysfunction as a mechanism of ICB resistance in mice and suggest potential prognostic utility of assessing IFN-related genes in patients with TNBC receiving ICB therapy.Significance:These data demonstrate for the first time that age determines the T cell–inflamed phenotype in TNBC and affects response to ICB in mice. Evaluating IFN-related genes from tumor genomic data may aid identification of patients for whom combination therapy including an IFN pathway activator with ICB may be required.This article is highlighted in the In This Issue feature, p. 1143

RASAL2 Expression/Promoter Methylation Analysis and Behavior of Various Breast Cancer Cell Lines.

PDF file - 214K, The LBC environment promotes pro-angiogenic cytokine enrichment in circulating platelets but does not significantly affect phospho-STAT3 levels in BMCs

PDF file - 51K, EGF and IGF-1 Together Modulate Tumorogenicity of Responder Cells in vitro.

PDF file 194K, Senescence-associated beta-galactosidase staining of BJ fibroblasts and treatment of HaCaT-CBR cells with the p38MAPK small-molecule inhibitor SB203580

PDF file - 125K, Supplemental Table 1: PCR Oligonucleotide Primers. Supplemental Table 2: Antibodies.

PDF file - 490K, Images used for quantification of responding tumor vessel density under indicated systemic environments; analysis reveals enhanced vascularization in the instigating LBC environment

PDF file - 174K, Activation of EGF and IGF1 Receptors.

DAB2IP Expression in a Panel of MMTVNeu/RASAL2-/- Tumors.

Background: Although immune checkpoint inhibitors (ICI) are most widely used in PD-L1 positive, metastatic triple-negative breast cancer (TNBC), the clinical utility of ICI in other tumor subtypes and patient populations is not well defined. In this study, we measured infiltration of multiple immune cell populations in primary and metastatic HR+ tumors to infer potential benefit from ICI. We investigated how risk stratifying features such as pathologic grade and Oncotype Dx scores affect immune cell infiltration across subtypes. Lastly, we compared immune cell infiltration in breast tumors from younger versus older women, as our pre-clinical studies demonstrated differential enrichment of immune response pathways by age and association with improved response to ICI. Methods: ImmunoProfile, a multiplexed immunofluorescence (IF) assay, was performed on FFPE tissue from 167 archived breast cancer tumors from the Dana-Farber Cancer Institute (HR+/HER2- n=124, HER2+ n=21, TNBC n=22). Unmatched samples were obtained from either primary breast (HR+/HER2- n=104, HER2+ n=13, TNBC n=15) or metastatic core biopsies. IF staining signal was measured for T cell markers CD8 and co-localized CD8/PD-1, T regulatory cell marker FOXP3, PD-L1 and PD1 expression on tumor cells as well as PD-L1 expression on inflammatory cells. Inflammatory, tumoral (TPS) and combined inflammatory and tumor (CPS) pathologic PD-L1 scores were calculated. PD-L1 staining was also assessed by IHC (E1L3N) staining and compared to multiplexed IF stains. IF staining signal (Vectra analyzed, log2+1 transformed) was analyzed against clinical and pathologic variables, including tumor subtype, primary versus metastatic status, pathologic grade, and Oncotype Dx score, using non-parametric statistical comparisons. Given the decline in adaptive immunity that occurs with normal physiological aging and our own group’s previous reports, we also explored whether immune cell staining signal associated with age. Results: TPS, CPS, and inflammatory PD-L1 scores strongly correlated between IHC and IF staining methods (Spearman r = 0.660, 0.683, 0.638 respectively, p < 2.2e-16). When HR+/HER2- (n=124), HER2+ (n=21) and TNBC (n=22) cases were analyzed separately, there was no statistical difference between unmatched primary and metastatic cases in any of the analyzed immune cell population stains for any subtype. When all primary cases were analyzed, TNBC showed higher CD8, CD8/PD1, PD1 (all p < 0.05), and FOXP3 staining as well as IF CPS, TPS and inflammatory PD-L1 scores (all p < 0.005) than HR+ cases. HR+/HER2- cases from women < age 65 (n=56) showed higher intra-tumoral CD8 (p=0.039), PD1 (p=0.043), and CD8/PD1 (p=0.022) than cases from women > age 65 (n=38). There was a statistically significant step-wise increase in IF staining of all immune cell populations with increasing pathologic grade (I, II, III) as well as Oncotype Dx scores (< 11, 11-25, >25) among primary HR+/HER2- cases (n=74). Conclusions: Strong correlations between PD-L1 scores based on IF and IHC methods suggest clinical utility for multiplexed IF immune cell staining in therapeutic planning. ImmunoProfile demonstrated the previously known pattern that primary TNBC contains higher immune cell infiltration than HR+ or HER2+ tumors. Women < age 65 with HR+ cancer showed higher levels of CD8 and PD1 stained populations, suggesting there may be greater utility of ICI in younger patients. Lastly, the association of higher pathologic grade and Oncotype Dx score with increasing immune cell infiltration supports exploration of ICI in biologically unfavorable primary HR+ cancers. Citation Format: Tess A. O'Meara, Tanya E. Keenan, Adrienne G. Waks, Kristen D. Felt, Bijaya Sharma, Scott Rodig, Melissa Hughes, Nancy U. Lin, Judith Agudo, Jennifer L. Guerriero, Sandra S. McAllister, Elizabeth Mittendorf, Sara Tolaney. Multiplexed immunofluorescence staining of intra-tumoral immune cell populations and associations with immunohistochemical, clinical, and pathologic variables in breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-04-05.

The presence of disseminated tumor cells in breast cancer patient bone marrow aspirates predicts decreased recurrence-free survival. Although it is appreciated that physiologic, pathologic, and therapeutic conditions impact hematopoiesis, it remains unclear whether targeting hematopoiesis presents opportunities for limiting bone metastasis. Using preclinical breast cancer models, we discovered that marrow from mice treated with the bisphosphonate zoledronic acid (ZA) are metastasis-suppressive. Specifically, ZA modulated hematopoietic myeloid/osteoclast progenitor cell (M/OCP) lineage potential to activate metastasis-suppressive activity. Granulocyte-colony stimulating factor (G-CSF) promoted ZA resistance by redirecting M/OCP differentiation. We identified M/OCP and bone marrow transcriptional programs associated with metastasis suppression and ZA resistance. Analysis of patient blood samples taken at randomization revealed that women with high-plasma G-CSF experienced significantly worse outcome with adjuvant ZA than those with lower G-CSF levels. Our findings support discovery of therapeutic strategies to direct M/OCP lineage potential and biomarkers that stratify responses in patients at risk of recurrence.Significance: Bone marrow myeloid/osteoclast progenitor cell lineage potential has a profound impact on breast cancer bone metastasis and can be modulated by G-CSF and bone-targeting agents. Cancer Res; 78(18); 5300–14. ©2018 AACR.

Supplementary Figures and Table Legends to support primary figures

Gene set enrichment analyses showing effects of TNBC on tumor-promoting BMCs of young and old mice

Supplemental Figure 1 shows the tumor masses of the subcutaneous tumors shown in Figure 1, as well as the flow cytometry gating strategy of M/OCP populations and the osteoclast differentiation assay experimental schematic. Supplemental Figure 2 shows representative images and the flow cytometry gating strategy for the in vitro osteoclast differentiation assay demonstrated in Figure 2. Supplemental Figure 3 shows that ZA inhibits breast cancer metastasis in a manner that is counteracted by G-CSF in intratibial injections, subcutaneous bone marrow functional assays, and with intracardiac injections of the B1 cell line following pre-treatment with Ctl or ZA and/or recombinant G-CSF. Supplemental Figure 4 shows the tumor masses of the subcutaneous tumors shown in Figure 4, as well as the plasma ALP and NTX concentrations in nude mice with and without ZA treatment. Supplemental Figure 5 shows the representative images and quantifications of the in vitro osteoclast differentiation assay with whole bone marrow from Ctl- or ZA- donors. Supplemental Figure 6 shows the volcano plots, heatmaps of differentially expressed genes, and gene ontology categories for the RNA-sequencing analysis of whole bone marrow from mice treated with Ctl, ZA, G-CSF, or ZA+G-CSF. Supplemental Figure 7 shows the determination of cut point analyses for the plasma G-CSF concentrations and patient outcome data shown in Figure 7.

Functionally enriched gene ontology (GO) terms and differentially enriched genes for the RNA-sequencing tables and heatmaps included in the manuscript text.

Supplementary figure 2. Aspirin treatment results in activation of AMPK and decrease in mTORC1 signaling.

Despite the high incidence of oncogenic mutations in PIK3CA, the gene encoding the catalytic subunit of PI3K, PI3K inhibitors have yielded little clinical benefit for breast cancer patients. Recent epidemiologic studies have suggested a therapeutic benefit from aspirin intake in cancers harboring oncogenic PIK3CA. Here, we show that mutant PIK3CA-expressing breast cancer cells have greater sensitivity to aspirin-mediated growth suppression than their wild-type counterparts. Aspirin decreased viability and anchorage-independent growth of mutant PIK3CA breast cancer cells independently of its effects on COX-2 and NF-κB. We ascribed the effects of aspirin to AMP-activated protein kinase (AMPK) activation, mTORC1 inhibition, and autophagy induction. In vivo, oncogenic PIK3CA-driven mouse mammary tumors treated daily with aspirin resulted in decreased tumor growth kinetics, whereas combination therapy of aspirin and a PI3K inhibitor further attenuated tumor growth. Our study supports the evaluation of aspirin and PI3K pathway inhibitors as a combination therapy for targeting breast cancer. Cancer Res; 77(3); 790–801. ©2016 AACR.

Controls and results from additional experiments to support primary data

PCR primer sequences

Detailed information for all antibodies

Supplementary figure 1. Expression of mutant PIK3CA increases COX-2 expression and IKKbeta/NF-KB signaling.

Detailed information for controls and experiments to support primary data