Metastatic breast cancer remains a significant source of mortality amongst breast cancer patients and is generally considered incurable in part due to the difficulty in detection of early micro-metastases. The pre-metastatic niche (PMN) is a tissue microenvironment that has undergone changes to support the colonization and growth of circulating tumor cells, a key component of which is the myeloid-derived suppressor cell (MDSC). Therefore, the MDSC has been identified as a potential biomarker for PMN formation, the detection of which would enable clinicians to proactively treat metastases. However, there is currently no technology capable of the in situ detection of MDSCs available in the clinic. Here, we propose the use of shortwave infrared-emitting nanoprobes for the tracking of MDSCs and identification of the PMN. Our rare-earth albumin nanocomposites (ReANCs) are engineered to bind the Gr-1 surface marker of murine MDSCs. When delivered intravenously in murine models of breast cancer with high rates of metastasis, the targeted ReANCs demonstrated an increase in localization to the lungs in comparison to control ReANCs. However, no difference was seen in the model with slower rates of metastasis. This highlights the potential utility of MDSC-targeted nanoprobes to assess PMN development and prognosticate disease progression.

Abstract Although triple-negative breast cancer (TNBC) can be treated with anti-PD-1 checkpoint immunotherapy in combination with chemotherapy, there remains a challenge in effectively monitoring therapeutic responses. Current non-invasive clinical imaging tools to evaluate response to treatment are reliant upon measurements of tumor volume and may fail to distinguish true progression from increased immune cell infiltration. Invasive biopsy sampling and immunohistochemistry (IHC) can elucidate changes in the immune landscape of treated tumors, but these methods are not conducive to providing real-time information. This study presents shortwave infrared (SWIR) imaging as a potential tool to detect treatment-induced cytotoxic T lymphocyte (CTL) infiltration non-invasively and in real time using rare earth metal-doped nanoparticles encapsulated in human serum albumin nanocomposites (ReANCs). ReANCs were chemically conjugated with anti-CD8α antibodies as targeting ligands to facilitate binding of the nanoprobes to CTLs with high specificity in a syngeneic mouse model of breast cancer. After treating the mice with combination anti-PD-1 and doxorubicin, volumetric analysis of the mammary fat pad tumors did not show any significant impact of treatment compared to single treatment and untreated control mice. However, increased CTL infiltration in the tumors of mice that received combination treatment was detected by in vivo SWIR imaging. CTL infiltration was validated with ex vivo IHC staining, and a monotonic relationship was observed between SWIR fluorescence and CD8 positivity. IHC staining of other immune markers, including CD45, CD3, CD4, and PD-L1, showed that combination treatment may influence in the expression of these markers, presenting additional targets that could be imaged with ReANCs in the future. In conclusion, the increase in SWIR signal from CD8-targeted ReANCs in tumors treated with combination immunotherapy and chemotherapy and the relationship with IHC staining highlight the ability to use SWIR imaging for non-invasive assessment of changes in immune dynamics following treatment. Citation Format: Jay V. Shah, Jake N. Siebert, Xinyu Zhao, Shuqing He, Richard E. Riman, Mei Chee Tan, Mark C. Pierce, Edmund C. Lattime, Vidya Ganapathy, Prabhas V. Moghe. Non-invasive shortwave infrared imaging of cytotoxic T lymphocyte infiltration for monitoring responses to combination immunotherapy and chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4178.

Checkpoint immunotherapy has made great strides in the treatment of solid tumors, but many patients do not respond to immune checkpoint inhibitors. Identification of tumor‐infiltrating cytotoxic T cells (CTLs) has the potential to stratify patients and monitor immunotherapy responses. In this study, the design of cluster of differentiation (CD8 + ) T cell‐targeted nanoprobes that emit shortwave infrared (SWIR) light in the second tissue‐transparent window for noninvasive, real‐time imaging of CTLs in murine models of breast cancer is presented. SWIR‐emitting rare‐earth nanoparticles encapsulated in human serum albumin are conjugated with anti‐CD8α to target CTLs with high specificity. CTL targeting is validated in vitro through binding of nanoprobes to primary mouse CTLs. The potential for the use of SWIR fluorescence intensity to determine CTL presence is validated in two syngeneic mammary fat pad tumor models, EMT6 and 4T1, which differ in immune infiltration. SWIR imaging using CD8‐targeted nanoprobes successfully identifies the presence of CTLs in the more immunogenic EMT6 model, while imaging confirms the lack of substantial immune infiltration in the nonimmunogenic 4T1 model. In this work, the opportunity for SWIR imaging using CD8‐targeted nanoprobes to assess CTL infiltration in tumors for the stratification and monitoring of responders to checkpoint immunotherapy is highlighted.

BackgroundLate-stage diagnosis of ovarian cancer, a disease that originates in the ovaries and spreads to the peritoneal cavity, lowers 5-year survival rate from 90% to 30%. Early screening tools that can: i) detect with high specificity and sensitivity before conventional tools such as transvaginal ultrasound and CA-125, ii) use non-invasive sampling methods and iii) longitudinally significantly increase survival rates in ovarian cancer are needed. Studies that employ blood-based screening tools using circulating tumor-cells, -DNA, and most recently tumor-derived small extracellular vesicles (sEVs) have shown promise in non-invasive detection of cancer before standard of care. Our findings in this study show the promise of a sEV-derived signature as a non-invasive longitudinal screening tool in ovarian cancer. MethodsHuman serum samples as well as plasma and ascites from a mouse model of ovarian cancer were collected at various disease stages. Small extracellular vesicles (sEVs) were extracted using a commercially available kit. RNA was isolated from lysed sEVs, and quantitative RT-PCR was performed to identify specific metastatic gene expression. ConclusionThis paper highlights the potential of sEVs in monitoring ovarian cancer progression and metastatic development. We identified a 7-gene panel in sEVs derived from plasma, serum, and ascites that overlapped with an established metastatic ovarian carcinoma signature. We found the 7-gene panel to be differentially expressed with tumor development and metastatic spread in a mouse model of ovarian cancer. The most notable finding was a significant change in the ascites-derived sEV gene signature that overlapped with that of the plasma-derived sEV signature at varying stages of disease progression. While there were quantifiable changes in genes from the 7-gene panel in serum-derived sEVs from ovarian cancer patients, we were unable to establish a definitive signature due to low sample number. Taken together our findings show that differential expression of metastatic genes derived from circulating sEVs present a minimally invasive screening tool for ovarian cancer detection and longitudinal monitoring of molecular changes associated with progression and metastatic spread.

Abstract Checkpoint immunotherapy, through the reversal of tumor-mediated inactivation of the immune system, has shown promise in the treatment of several types of cancer. This has culminated in the approval of seven immune checkpoint inhibitors (ICIs). However, only a small population of patients respond to these drugs. Because of the physical and economic burden of ICIs on the patient, there is a critical need to identify biomarkers that can inform on the potential response to ICIs. The presence of tumor infiltrating lymphocytes (TILs) has demonstrated good prognostic value in determining if a patient should receive ICIs. Current clinical methods to assess TILs involve invasive biopsies and immunohistochemistry, which suffer from intratumoral heterogeneity, observer variability, and a lack of real-time feedback. Here, we report on near infrared light excitable rare earth metal-based nanoparticles, termed rare earth albumin nanocomposites (ReANCs), that emit shortwave infrared (SWIR) light, allowing for deep tissue imaging and high signal-to-noise ratios compared to visible or near infrared fluorescence probes. Tumor-targeted ReANCs have been previously employed to monitor tumor progression and response to chemotherapy in mouse models of breast cancer metastasis. In this study, to target CD3+ T cells, ReANCs were conjugated using the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to a peptide derived from the sequence of the CD3-ϵ receptor sub-unit. Target specific binding was validated by flow cytometry as a measure of increased uptake of peptide-conjugated ReANCs by Jurkat cells. To specifically target cytotoxic T lymphocytes, we employed the fragment antigen binding (Fab) derived from enzymatic digestion of a CD8 antibody (clone 53-6.7) with papain. The Fab fragments were conjugated to ReANCs with sulfo-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC). Conjugation was confirmed by non-reducing gel electrophoresis and high performance liquid chromatography (HPLC). A loading efficiency of approximately 60% was achieved. Target specific binding was validated by flow cytometry as a measure of increased uptake of Fab-conjugated ReANCs by T cells isolated from splenocytes. We generated a metric for measuring immune burden around tumor spheroids by pre-labeling T cells with ReANCs and co-culturing them with tumor cell spheroids in vitro. Imaging of T cells with CD3 and CD8-targeted ReANCs provides a basis for future in vivo small animal imaging studies where we will investigate the potential of this technology to track immune cells in relation to a tumor in real time. Metrics of immune cell imaging will then inform on the potential of immunotherapy and monitor response to treatment in a longitudinal study. Citation Format: Jay V. Shah, Jake N. Siebert, Amber Gonda, Rahul Pemmaraju, Shashank Kosuri, Carolina Bobadilla Mendez, Xinyu Zhao, Shuqing He, Richard E. Riman, Mei Chee Tan, Mark C. Pierce, Edmund C. Lattime, Prabhas V. Moghe, Vidya Ganapathy. Rare earth albumin nanoparticles engineered to target cytotoxic T cells to evaluate response to immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2802.

Abstract Early diagnosis and effective tumor monitoring can significantly alter clinical outcomes of ovarian cancer patients. Innovative tools are needed to enhance the sensitivity and specificity of current monitoring modalities. Extracellular vesicles, or exosomes, have shown to be promising conduits of diagnostic biomarkers to aid in tumor detection as evidenced by Exosome Diagnostics' new ExoDx Prostate (IntelliScore) test that uses exosomal markers to differentiate between benign prostate disease and early cancer (Tutrone, R., Donovan, M.J., Torkler, P. et al. Clinical utility of the exosome based ExoDx Prostate(IntelliScore) EPI test in men presenting for initial Biopsy with a PSA 2-10 ng/mL. Prostate Cancer Prostatic Dis 23, 607-614 (2020)). The potential of these vesicles however goes beyond simple diagnostic power of cancer detection. Due to the onco-specific contents packaged and the minimally invasive, low risk accessibility, exosomes have the capacity to be used as longitudinal monitoring tools to characterize early molecular changes at all stages of the disease. We hypothesized that the dynamicity of ovarian tumors during progression and metastatic development is reflected in exosomes. In order to test this we isolated exosomes and used qPCR to analyze exosomal gene signatures from a mouse model of ovarian cancer. SKOV3 ovarian cancer tumor cells were injected into mice and allowed to grow for 3 weeks. Plasma was collected from mice at 5-7 day increments and exosomes were extracted. Multiple established metastatic genes in ovarian cancer were evaluated and 4 genes, Lox, THBS1, TIMP3, and β-actin, were found to be differentially expressed in correlation with 3 translationally pertinent assessments: presence or absence of tumors, levels of metastatic burden, and longitudinal tumor progression. Gene expression patterns were compared with exosomal gene signatures extracted from human ovarian patient plasma and found to express similar patterns. These results support the diagnostic potential of using exosomal genetic signatures to detect early metastatic development and to facilitate longitudinal tracking of tumor progression. Citation Format: Amber Gonda, Jay V. Shah, Jake N. Siebert, Nanxia Zhao, Mi Jung Kwon, Prabhas V. Moghe, Nicola Francis, Vidya Ganapathy. Exosome gene signatures characterize metastatic dynamicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2831.

Fluorescence-guided surgery (FGS) is an emerging technique for tissue visualization during surgical procedures. Structures of interest are labeled with exogenous probes whose fluorescent emissions are acquired and viewed in real-time with optical imaging systems. This study investigated rare-earth-doped albumin-encapsulated nanocomposites (REANCs) as short-wave infrared emitting contrast agents for FGS. Experiments were conducted using an animal model of 4T1 breast cancer. The signal-to-background ratio (SBR) obtained with REANCs was compared to values obtained using indocyanine green (ICG), a near-infrared dye used in clinical practice. Prior to resection, the SBR for tumors following intratumoral administration of REANCs was significantly higher than for tumors injected with ICG. Following FGS, evaluation of fluorescence intensity levels in excised tumors and at the surgical bed demonstrated higher contrast between tissues at these sites with REANC contrast than ICG. REANCs also demonstrated excellent photostability over 2 hours of continuous illumination, as well as the ability to perform FGS under ambient lighting, establishing these nanocomposites as a promising contrast agent for FGS applications.