Tumor Periphery Research Articles

Abstract 4588: Vascular regulation of T-cell exclusion in melanoma

Abstract T-cell position in the tumor microenvironment determines the probability of target encounter and tumor killing. CD8+ T-cell exclusion from the tumor parenchyma is associated with poor response to immunotherapy and yet the biology that underpins this distinct pattern remains unclear. Here we show that the vascular destabilizing factor angiopoietin-2 (ANGPT2) causes compromised vascular integrity in the tumor periphery, leading to impaired T-cell infiltration within tumor. We analyzed the spatial regulation of ANGPT2 in whole tumor cross-sections and its impact on CD8+ T-cell position, vascular integrity, and response to immunotherapy using syngeneic murine melanomas. We found that T-cell exclusion was associated with peritumoral expression of ANGPT2 and elevated vascular leakage in human and murine melanomas. Both pharmacological and genetic blockade of ANGPT2 promoted CD8+ T-cell infiltration to the tumor core, exerting antitumor effects. Importantly, the reversal of T-cell exclusion following ANGPT2 blockade not only enhanced response to anti-PD-1 therapy in immunogenic, therapy responsive mouse melanomas, but also rendered non-responsive tumors susceptible. Therapeutic response after ANGPT2 blockade, driven by improved CD8+ T-cell infiltration, coincided with TIE2 signaling activation and increased vascular integrity in the tumor periphery, indicating that maintenance of vascular integrity within tumors is necessary for robust response to immunotherapy. These data highlight ANGPT2/TIE2 signaling as a key mediator of T-cell exclusion and a promising target to potentiate checkpoint inhibitor efficacy in melanoma. Citation Format: Ha-Ram Park, Anahita Shiva, Portia Cummings, Richard Carvajal, Gavin Thurston, Joon-Yong An, Amanda W. Lund, Hee Won Yang, Minah Kim. Vascular regulation of T-cell exclusion in melanoma. [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 4588.

Abstract 4614: Investigating lymphatic vessel remodeling and anti-tumor immunity in pancreatic cancer using tumor-on-chip and mouse models

Abstract Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest tumors in humans. PDAC has an immunosuppressive tumor microenvironment, constituting for poor immunotherapy outcomes. Lymphatic vessels (LVs) transport leukocytes and antigens from tumor peripheries to draining lymph nodes (dLNs) for activation of anti-tumor immune responses. It is believed that impairment of the lymphatic function may result in delayed and ineffective immune activation against tumors. In this study, we created a three-dimensional (3D) tumor-on-chip model to examine phenotypical and functional changes in 3D engineered LVs, composed of human primary lymphatic endothelial cells (LECs) in co-culture with PDAC cells. Our in vitro studies revealed that PDAC cells induce lymphangiogenesis and tighten cell-cell junctions in LECs, forming “zipper-like” LEC junctions, compared to control LECs with “button-like” junctions. We introduced 70kDa dextran into the LV lumens and showed PDAC-induced zippering of junctions decreased vessel permeability. These results led us to hypothesize that altered LEC junctions may affect fluid drainage into the LVs in vivo. We inoculated PDAC cells into the mouse ear pinnae and performed whole-mount immunostaining and lymphatic drainage experiment by injecting Evans blue into the ears and measured the remained Evans blue in the ears. In the ear experiments, inoculated PDAC cells affected host LVs with originally button-like junctions to become zipper-like and impede lymphatic drainage. To identify a potential signaling pathway that may be responsible for LEC junction zippering, we used phospho-kinase arrays and discovered that PDAC cells induced LECs to increase phosphorylation of GSK3-β, JNK kinases, and enhanced the total protein level of β-catenin, implying that PDAC cells turned on the WNT signaling in LECs. In preliminary screening, we treated LECs co-cultured with PDAC cells with WNT-C59, an WNT inhibitor that blocks secretion of all WNT ligands, which resulted in decreased number of LV sprouting and less contractile actin-cytoskeleton in LECs. It is critical to know which molecules were secreted by PDAC cells and influenced the WNT signaling in LECs. With that in mind, we plan to perform mass spectrometry with PDAC-conditioned media to identify PDAC-specific secretome; and run RNA-seq with LECs isolated from mouse ears with or without PDAC to investigate molecular basis of the WNT activation in LECs and LV remodeling. Finally, we plan to examine the egress of leukocytes from primary PDAC tumor site, T cell activation in tumor-dLNs, and PDAC tumor growth with or without WNT inhibition in Kaeda mice. The identity and function of PDAC-egressed immune cells, and those which stayed within the primary tumor will enhance our understanding of the roles of LVs in orchestrating anti-tumor immunity in PDAC. Citation Format: Anna Maria Kolarzyk, Conor Loy, Renhao Lu, Iwijn De Vlaminck, Deborah Fowell, Esak Lee. Investigating lymphatic vessel remodeling and anti-tumor immunity in pancreatic cancer using tumor-on-chip and mouse models. [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 4614.

Cathepsin protease expression in infiltrative soft tissue sarcomas: cathepsin-K correlates with infiltrative tumor growth and clinical outcomes.

Cathepsin proteases, activated in the lysosomes, are upregulated in many cancers. Intraoperative detection systems of microscopic residual tumor using cathepsin-mediated release of fluorescent nanoparticles may guide surgical excisions to improve local control. We sought to define the genetic and proteomic expression of cathepsins and their clinicopathological correlates in myxofibrosarcoma and undifferentiated pleomorphic sarcoma (UPS)-soft tissue sarcomas with high rates of positive resection margins and local recurrence-and to establish a cellular justification for cathepsin-dependent systems to identify residual cancer in the resection bed. Real-time quantitative polymerase chain reaction analysis of 58 fresh-frozen tumor specimens revealed that 56 (97%) had elevated mRNA expression of ≥1 cathepsin, including cathepsin-B (79%), cathepsin-K (59%), cathepsin-L (71%), and -S (71%). Immunohistochemical analysis of these fresh-frozen specimens revealed that 98% of tumors were positive for one or more of cathepsin-B (85%), cathepsin-K (50%), cathepsin-L (63%), and -S (10%). Strong cathepsin-K expression was associated with greater risks of local recurrence (hazard ratio, 3.78; p=0.044) and disease-specific mortality (hazard ratio, 3.70; p=0.025). Immunohistochemical analysis of 33 formalin-fixed paraffin-embedded block samples revealed that 97% were positive for cathepsin-B (88%), cathepsin-K (76%), cathepsin-L (52%), or -S (52%) at the tumor periphery; cathepsin-K positivity correlated with a radiographic tail-like sign (p=0.004) and microscopic infiltrative growth (p=0.020). We conclude that cathepsins are broadly overexpressed in myxofibrosarcoma and UPS, and cathepsin-K may be an immunohistochemical marker of local infiltration and poorer prognosis that could be used to guide precision surgery.

Senescent Tumor Cells Are Frequently Present at the Invasion Front: Implications for Improving Disease Control in Patients with Locally Advanced Prostate Cancer

Introduction: Local tumor invasion is a critical factor for the outcome of men with prostate cancer. In particular, seminal vesicle invasion (SVI) has been reported to be associated with a more unfavorable prognosis. A better understanding of the functional state of invading prostate cancer cells is crucial to develop novel therapeutic strategies for patients with locally advanced disease. Methods: The prognostic impact of local tumor progression was ascertained in over 1,000 men with prostate cancer. Prostate cancer specimens were stained by double-immunohistochemistry for the proliferation marker Ki-67 and the senescence marker p16^INK4A. The migratory properties of senescent prostate cancer cells were analyzed in vitro using a wound healing assay and immunofluorescence microscopy for p16^INK4A. Results: We confirm the notion that patients with SVI have a more unfavorable prognosis than patients with extraprostatic extension alone. Surprisingly, we found that the tumor invasion front frequently harbors p16^INK4A-positive and Ki-67-negative, i.e., senescent, tumor cells. While the intraprostatic tumor periphery was a hotspot for both proliferation and expression of p16^INK4A, the area of SVI showed less proliferative activity but was at the same time a hotspot of cells with increased nuclear p16^INK4A expression. Senescence was associated with an accelerated migration of prostate cancer cells in vitro. Conclusion: This proof-of-concept study shows that invading prostate cancer cells frequently show signs of cellular senescence. This finding may open new avenues for neoadjuvant and adjuvant treatment concepts in men with locally advanced prostate cancer.

State-dependent evolutionary models reveal modes of solid tumour growth

Spatial properties of tumour growth have profound implications for cancer progression, therapeutic resistance and metastasis. Yet, how spatial position governs tumour cell division remains difficult to evaluate in clinical tumours. Here, we demonstrate that faster division on the tumour periphery leaves characteristic genetic patterns, which become evident when a phylogenetic tree is reconstructed from spatially sampled cells. Namely, rapidly dividing peripheral lineages branch more extensively and acquire more mutations than slower-dividing centre lineages. We develop a Bayesian state-dependent evolutionary phylodynamic model (SDevo) that quantifies these patterns to infer the differential division rates between peripheral and central cells. We demonstrate that this approach accurately infers spatially varying birth rates of simulated tumours across a range of growth conditions and sampling strategies. We then show that SDevo outperforms state-of-the-art, non-cancer multi-state phylodynamic methods that ignore differential sequence evolution. Finally, we apply SDevo to single-time-point, multi-region sequencing data from clinical hepatocellular carcinomas and find evidence of a three- to six-times-higher division rate on the tumour edge. With the increasing availability of high-resolution, multi-region sequencing, we anticipate that SDevo will be useful in interrogating spatial growth restrictions and could be extended to model non-spatial factors that influence tumour progression.

Abstract P1-05-17: Radiomic models based on dynamic contrast-enhanced magnetic resonance imaging predict the immunophenotype reflecting spatial distribution of CD8+ tumor-infiltrating lymphocytes in breast cancer

Abstract Purpose: This study aimed to develop noninvasive magnetic resonance imaging (MRI)-based radiomic models to predict immunophenotypes of breast cancer. Methods: A total of 182 breast cancer patients who underwent upfront surgery were analyzed, divided into training (n = 137) and validation (n = 45) cohorts. Dynamic contrast-enhanced (DCE)-MRI was acquired, and immunophenotype was determined on the surgical tumor sections: immune-inflamed (high degree of CD8+ T cells infiltrated), -excluded (CD8+ T cells accumulated at invasive margin but not efficiently infiltrated in tumor bed), and -desert (CD8+ T cells absent within tumor and at margins). Based on 833 radiomic features extracted after manual delineation, the least absolute shrinkage and selection operator method was used to build radiomic models. Results: Our radiomic models from the whole tumor sections showed moderate performance in predicting the immune-inflamed versus non-inflamed tumors, showing the highest AUC values, of 0.659 and 0.671 for training and validation cohorts, respectively. Combining the models, much improved predictability was observed, with AUC values of 0.973 and 0.985 for training and validation cohorts, respectively. Other radiomic features from tumor periphery data discriminated immune-excluded versus immune-desert status, with AUC values of 0.993 and 0.984 for training and validation cohorts, respectively. The combined models were also applicable to predicting immunophenotype for different molecular subtypes, with AUC values ≥ 0.867. Conclusions: By integrating the immunohistochemistry profiles, we established MRI-derived radiomic models to predict the detailed immunophenotype of breast cancer. This study suggests the feasibility of noninvasive assessment of tumor immune status in real-world clinics. Citation Format: Seung Hyuck Jeon, So-Woon Kim, Mirinae Seo, Yu Jin Lim. Radiomic models based on dynamic contrast-enhanced magnetic resonance imaging predict the immunophenotype reflecting spatial distribution of CD8+ tumor-infiltrating lymphocytes in breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-05-17.

An agonistic anti-Tie2 antibody suppresses the normal-to-tumor vascular transition in the glioblastoma invasion zone

Tumor progression is intimately associated with the vasculature, as tumor proliferation induces angiogenesis and tumor cells metastasize to distant organs via blood vessels. However, whether tumor invasion is associated with blood vessels remains unknown. As glioblastoma (GBM) is featured by aggressive invasion and vascular abnormalities, we characterized the onset of vascular remodeling in the diffuse tumor infiltrating zone by establishing new spontaneous GBM models with robust invasion capacity. Normal brain vessels underwent a gradual transition to severely impaired tumor vessels at the GBM periphery over several days. Increasing vasodilation from the tumor periphery to the tumor core was also found in human GBM. The levels of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) showed a spatial correlation with the extent of vascular abnormalities spanning the tumor-invading zone. Blockade of VEGFR2 suppressed vascular remodeling at the tumor periphery, confirming the role of VEGF-VEGFR2 signaling in the invasion-associated vascular transition. As angiopoietin-2 (ANGPT2) was expressed in only a portion of the central tumor vessels, we developed a ligand-independent tunica interna endothelial cell kinase 2 (Tie2)-activating antibody that can result in Tie2 phosphorylation in vivo. This agonistic anti-Tie2 antibody effectively normalized the vasculature in both the tumor periphery and tumor center, similar to the effects of VEGFR2 blockade. Mechanistically, this antibody-based Tie2 activation induced VE-PTP-mediated VEGFR2 dephosphorylation in vivo. Thus, our study reveals that the normal-to-tumor vascular transition is spatiotemporally associated with GBM invasion and may be controlled by Tie2 activation via a novel mechanism of action.

Volumetric imaging of optically cleared and fluorescently labeled animal tissue (VIOLA) for quantifying the 3D biodistribution of nanoparticles at cellular resolution in tumor tissue.

Nanoparticle (NP) technology holds significant promise to mediate targeted drug delivery to specific organs in the body. Understanding the 3D biodistribution of NPs in heterogeneous environments such as the tumor tissue can provide crucial information on efficacy, safety and potential clinical outcomes. Here we present a novel end-to-end workflow, VIOLA, which makes use of tissue clearing methodology in conjunction with high resolution imaging and advanced 3D image processing to quantify the spatiotemporal 3D biodistribution of fluorescently labeled ACCURIN® NPs. Specifically, we investigate the spatiotemporal biodistribution of NPs in three different murine tumor models (CT26, EMT6, and KPC-GEM) of increasing complexity and translational relevance. We have developed new endpoints to characterize NP biodistribution at multiple length scales. Our observations reveal that the macroscale NP biodistribution is spatially heterogeneous and exhibits a gradient with relatively high accumulation at the tumor periphery that progressively decreases towards the tumor core in all the tumor models. Microscale analysis revealed that NP extravasation from blood vessels increases in a time dependent manner and plateaus at 72h post injection. Volumetric analysis and pharmacokinetic modeling of NP biodistribution in the vicinity of the blood vessels revealed that the local NP density exhibits a distance dependent spatiotemporal biodistribution which provide insights into the dynamics of NP extravasation in the tumor tissue. Our data represents a comprehensive analysis of NP biodistribution at multiple length scales in different tumor models providing unique insights into their spatiotemporal dynamics. Specifically, our results show that NPs exhibit a dynamic equilibrium with macroscale heterogeneity combined with microscale homogeneity.

Gold nanorods assisted photothermal therapy of bladder cancer in mice: A computational study on the effects of gold nanorods distribution at the centre, periphery, and surface of bladder cancer

Background and objectivesGold nanorod-assisted photothermal therapy (GNR-PTT) is a cancer treatment whereby GNRs incorporated into the tumour act as photo-absorbers to elevate the thermal destruction effect. In the case of bladder, there are few possible routes to target the tumour with GNRs, namely peri/intra-tumoural injection and intravesical instillation of GNRs. These two approaches lead to different GNR distribution inside the tumour and can affect the treatment outcome. MethodologyThe present study investigates the effects of heterogeneous GNR distribution in a typical setup of GNR-PTT. Three cases were considered. Case 1 considered the GNRs at the tumour centre, while Case 2 represents a hypothetical scenario where GNRs are distributed at the tumour periphery; these two cases represent intratumoural accumulation with different degree of GNR spread inside the tumour. Case 3 is achieved when GNRs target the exposed tumoural surface that is invading the bladder wall, when they are delivered by intravesical instillation. ResultsResults indicate that for a laser power of 0.6 W and GNR volume fraction of 0.01%, Case 2 and 3 were successful in achieving complete tumour eradication after 330 and 470 s of laser irradiation, respectively. Case 1 failed to form complete tumour damage when the GNRs are concentrated at the tumour centre but managed to produce complete tumour damage if the spread of GNRs is wider. Results from Case 2 also demonstrated a different heating profile from Case 1, suggesting that thermal ablation during GNR-PTT is dependant on the GNRs distribution inside the tumour. Case 3 shows similar results to Case 2 whereby gradual but uniform heating is observed. Cases 2 and 3 show that uniformly heating the tumour can reduce damage to the surrounding tissues. ConclusionsDifferent GNR distribution associated with the different methods of introducing GNRs to the bladder during GNR-PTT affect the treatment outcome of bladder cancer in mice. Insufficient spreading during intratumoural injection of GNRs can render the treatment ineffective, while administered via intravesical instillation. GNR distribution achieved through intravesical instillation present some advantages over intratumoural injection and is worthy of further exploration.

GPR4 in the pH-dependent migration of melanoma cells in the tumor microenvironment.

Due to its high metastatic potential, malignant melanoma is one of the deadliest skin cancers. In melanoma as well as in other cancers, acidification of the tumor microenvironment (=TME, inverse pH-gradient) is a well-known driver of tumor progression and metastasis. Membrane-bound receptors, such as the proton-sensitive GPCR (pH-GPCR) GPR4, are considered as potential initiators of the signalling cascades relevant to malignant transformation. In this study, we investigated the pH-dependent migration of GPR4 wildtype/overexpressing SK-Mel-28 cells using an impedance-based electrical wounding and migration assay and classical Boyden chamber experiments. Migration of GPR4 overexpressing SK-Mel-28 cells was enhanced in a range of pH6.5-7.5 as compared to controls in the impedance-based electrical wounding and migration assay. In Boyden chamber experiments, GPR4 overexpression only increased migration at pH7.5 in a Matrigel-free setup, but not at pH6.5. Results indicate that GPR4 is involved in the migration of melanoma cells, especially in the tumor periphery, and that this process is affected by pH in the TME.

Treatment of pancreatic cancer with irreversible electroporation and intratumoral CD40 antibody stimulates systemic immune responses that inhibit liver metastasis in an orthotopic model

BackgroundPancreatic cancer (PC) has a poor prognosis, and most patients present with either locally advanced or distant metastatic disease. Irreversible electroporation (IRE) is a non-thermal method of ablation used clinically...

Magnetic nanoparticle swarm with upstream motility and peritumor blood vessel crossing ability.

Micro-nano-robots show great potential and value for applications in targeted drug delivery; however, very few current studies have enabled micro-nano-robots to move against blood flow, and in addition, how micro-nano-robots can penetrate endothelial cells and enter tissues via vascular permeation remains unclear. Inspired by the bionics of dynamic aggregation in wild herring schools and transvascular permeation of leukocytes, we propose a novel drug delivery strategy where thousands of magnetic nanoparticles (MNPs) can be assembled into swarms under the guidance of a specially designed electromagnetic field. The vortex-like swarms of magnetic nanoparticles exhibit excellent stability, allowing them to withstand the impact of high-speed flow and move upstream along the vessel wall, stopping at the target location. When the vortex-like swarms encounter a tumor periphery without a continuous vessel wall, their rheological properties actively adhere them to the edges of the vascular endothelial gap, using their deformability to crawl through narrow intercellular gaps, enabling large-scale targeted drug delivery. This cluster of miniature nanorobots can be reshaped and reconfigured to perform a variety of tasks according to the environmental demands of the circulatory system, providing new solutions for a variety of biomedical field applications.

Autofluorescence Image-Guided Endoscopy in the Management of Upper Aerodigestive Tract Tumors.

At this juncture, autofluorescence and narrow-band imaging have resurfaced in the medicine arena in parallel with current technology advancement. The emergence of newly developed optical instrumentation in addition to the discovery of new fluorescence biomolecules have contributed to a refined management of diseases and tumors, especially in the management of upper aerodigestive tract tumors. The advancement in multispectral imaging and micro-endoscopy has also escalated the trends further in the setting of the management of this tumor, in order to gain not only the best treatment outcomes but also facilitate early tumor diagnosis. This includes the usage of autofluorescence endoscopy for screening, diagnosis and treatment of this tumor. This is crucial, as microtumoral deposit at the periphery of the gross tumor can be only assessed via an enhanced endoscopy and even more precisely with autofluorescence endoscopic techniques. Overall, with this new technique, optimum management can be achieved for these patients. Hence, the treatment outcomes can be improved and patients are able to attain better prognosis and survival.

In vivo morphological alterations of TAMs during KCa3.1 inhibition-by using in vivo two-photon time-lapse technology.

Tumor associated macrophages (TAMs) are the mostprevalent cells recruited in the tumor microenvironment (TME). Once recruited, TAMs acquire a pro-tumor phenotype characterized by a typical morphology: ameboid in the tumor core and with larger soma and thick branches in the tumor periphery. Targeting TAMs by reverting them to an anti-tumor phenotype is a promising strategy for cancer immunotherapy. Taking advantage of Cx3cr1GFP/WT heterozygous mice implanted with murine glioma GL261-RFP cells we investigated the role of Ca2+-activated K+ channel (KCa3.1) on the phenotypic shift of TAMs at the late stage of glioma growth through in vivo two-photon imaging. We demonstrated that TAMs respond promptly to KCa3.1 inhibition using a selective inhibitor of the channel (TRAM-34) in a time-dependent manner by boosting ramified projections attributable to a less hypertrophic phenotype in the tumor core. We also revealed a selective effect of drug treatment by reducing both glioma cells and TAMs in the tumor core with no interference with surrounding cells. Taken together, our data indicate a TRAM-34-dependent progressive morphological transformation of TAMs toward a ramified and anti-tumor phenotype, suggesting that the timing of KCa3.1 inhibition is a key point to allow beneficial effects on TAMs.

Incorporating cross-voxel exchange for the analysis of dynamic contrast-enhanced imaging data: pre-clinical results

Tumours exhibit abnormal interstitial structures and vasculature function often leading to impaired and heterogeneous drug delivery. The disproportionate spatial accumulation of a drug in the interstitium is determined by several microenvironmental properties (blood vessel distribution and permeability, gradients in the interstitial fluid pressure). Predictions of tumour perfusion are key determinants of drug delivery and responsiveness to therapy. Pharmacokinetic models allow for the quantification of tracer perfusion based on contrast enhancement measured with non-invasive imaging techniques. An advanced cross-voxel exchange model (CVXM) was recently developed to provide a comprehensive description of tracer extravasation as well as advection and diffusion based on cross-voxel tracer kinetics (Sinno et al 2021). Transport parameters were derived from DCE-MRI of twenty TS-415 human cervical carcinoma xenografts by using CVXM. Tracer velocity flows were measured at the tumour periphery (mean 1.78–5.82 μm.s−1) pushing the contrast outward towards normal tissue. These elevated velocity measures and extravasation rates explain the heterogeneous distribution of tracer across the tumour and its accumulation at the periphery. Significant values for diffusivity were deduced across the tumours (mean 152–499 μm2.s−1). CVXM resulted in generally smaller values for the extravasation parameter (mean 0.01–0.04 min−1) and extravascular extracellular volume fraction (mean 0.05–0.17) compared to the standard Tofts parameters, suggesting that Toft model underestimates the effects of inter-voxel exchange. The ratio of Tofts’ extravasation parameters over CVXM’s was significantly positively correlated to the cross-voxel diffusivity (P < 0.0001) and velocity (P = 0.0005). Tofts’ increased measurements were explained using Sinno et al (2021)’s theoretical work. Finally, a scan time of 15 min renders informative estimations of the transport parameters. However, a duration as low as 7.5 min is acceptable to recognize the spatial variation of transport parameters. The results demonstrate the potential of utilizing CVXM for determining metrics characterizing the exchange of tracer between the vasculature and the tumour tissue. Like for many earlier models, additional work is strongly recommended, in terms of validation, to develop more confidence in the results, motivating future laboratory work in this regard.

Supraorbital transciliary approach as primary route to fronto-basal high grade glioma resection with 5-Aminolevulinic Acid use: Technical note

BackgroundGliomas are diffuse intra-axial lesions, which can be accessed by multiple surgical corridors for a same location depending on the surgeon's preference. 5-Aminolevulinic Acid use facilitates the extend of resection in case of high-grade gliomas, especially when differentiating normal brain from tumor periphery is challenging. MethodsComplete resection of glioblastoma via a supraorbital transciliary approach with 5-Aminolevulinic Acid use was performed without any complications, as demonstrated on postoperative MRI. ResultsPatient was discharged on the third postoperative day. Wound follow-up shows good cosmetic result. Patient underwent concomitant chemo-radiation (Temozolomide- 60Gy) and adjuvant chemotherapy (Temozolomide). No tumor recurrence was noted at six months follow-up. ConclusionIn selected cases, supraorbital transciliary approach could be proposed as primary approach as it provides the advantage of full control over all the vasculo-nervous structures at skull base without the necessity of protective brain retractor use while the 5-Aminolevulinic Acid use allows a gross total resection.

Radiation Therapy Modulates Tumor Physical Characteristics to Reduce Intratumoral Pressure and Enhance Intratumoral Drug Delivery and Retention

PurposeHigh intratumoral pressure, caused by tumor cell-to-cell interactions, interstitial fluid pressure, and surrounding stromal composition, plays a substantial role in resistance to intratumoral drug delivery and distribution. Radiation therapy (XRT) is commonly administered in conjunction with different intratumoral drugs, but assessing how radiation can reduce pressure locally and help intratumoral drug administration and retention is important. Methods and Materials344SQ-parental or 344SQ-anti-programmed cell death protein 1-resistant lung adenocarcinoma cells were established in 129Sv/Ev mice, and irradiated with either 1 Gy × 2, 5 Gy × 3, 8 Gy × 3, 12 Gy × 3, or 20 Gy × 1. Intratumoral pressure was measured every 3 to 4 days after XRT. Contrast dye was injected into the tumors 3- and 6-days after XRT, and imaged to measure drug retention. ResultsIn the 344SQ-parental model, low-dose radiation (1 Gy × 2) created an early window of reduced intratumoral pressure 1 to 3 days after XRT compared with untreated control. High-dose stereotactic radiation (12 Gy × 3) reduced intratumoral pressure 3 to 12 days after XRT, and 20 Gy × 1 showed a delayed pressure reduction on day 12. Intermediate doses of radiation did not significantly affect intratumoral pressure. In the more aggressive 344SQ-anti-programmed cell death protein 1-resistant model, low-dose radiation reduced pressure 1 to 5 days after XRT, and 12 Gy × 3 reduced pressure 1 to 3 days after XRT. Moreover, both 1 Gy × 2 and 12 Gy × 3 significantly improved drug retention 3 days after XRT; however, there was no significance detected 6 days after XRT. Lastly, a histopathologic evaluation showed that 1 Gy × 2 reduced collagen deposition within the tumor, and 12 Gy × 3 led to more necrotic core and higher extracellular matrix formation in the tumor periphery. ConclusionsOptimized low-dose XRT, as well as higher stereotactic XRT regimen led to a reduction in intratumoral pressure and increased drug retention. The findings from this work can be readily translated into the clinic to enhance intratumoral injections of various anticancer agents.

Regulatory T Cells but Not Tumour-Infiltrating Lymphocytes Correlate with Tumour Invasion Depth in Basal Cell Carcinoma.

Basal cell carcinoma (BCC) is the most common skin malignancy worldwide. Current evidence suggests tumour-infiltrating lymphocytes (TILs) may influence the clinical outcomes of patients with BCC. The present study aimed to profile the infiltrative characteristics of stromal TILs and regulatory T cells (Treg cells) in the tumour centre (TC), tumour periphery (TP), and normal adjacent tissue (NAT) of BCC. A total of 111 samples from 43 cutaneous BCC cases were examined for TIL (CD3+) and Treg cell (FOXP3+/CD3+) expression using immunohistochemical techniques. The correlations of Treg cells with TILs, invasion depth, and tumour morphological risk were analysed. We identified a high mean proportion of Treg cells within the tumour (TC = 46.9%, TP = 56.1%, NAT = 51.8%) despite a relatively low median of TILs (TC = 12.7%, TP = 10.3%, NAT = 3.6%), supporting the classification of BCC as a cold tumour. A significant positive correlation was observed between the proportion of Treg cells and sTILs (ρ = 0.325, p &lt; 0.001), suggesting a predominant role of TILs in the infiltration of Treg cells. An inverse correlation discovered between Treg cells and tumour invasion depth (r = -0.36, p = 0.017) might indicate Treg cells' anti-tumour capacity in BCC.

Abstract C032: Combination CAF/myeloid targeting in PDAC

Abstract Immunotherapy has revolutionized clinical care for many cancers, yet these treatments fail to control disease in many patients and new strategies are needed to improve anti-tumor immunity and enhance response rates. There is great need for an increased understanding of the cellular crosstalk within tumors and the identification of stromal and immune populations involved in shaping the tumor microenvironment (TME). Local immunosuppression (LIS) is one of the striking hallmarks of Pancreatic Ductal Adenocarcinoma (PDAC), a disease that is highly resistant to existing immunotherapies. Oncogenic Kras activation in tumor cells promotes the invasion and proliferation of tumor-supporting stromal cells, while excluding cancer-targeted cytotoxic T cells. LIS is mediated by multiple subtypes of cancer-associated fibroblasts (CAFs) and myeloid cells resident within the tumor parenchyma. Multiple prior attempts to reverse LIS in PDAC by targeting individual stromal cell populations have been unsuccessful, alluding to the complexity of stromal crosstalk within the TME. The stromal diversity of PDAC complicates investigating paracrine cascades involving multiple cell types. To decipher diverse drug effects on altering the TME, we employ in vivo studies in mouse models recapitulating the human disease, as well as a novel tumor explant model that enables the short-term culture of intact human or murine PDAC. Importantly, PDAC explants maintain their histopathological architecture and cellular diversity over time. This medium-throughput platform allows for testing of multiple drugs and mechanistic hypotheses in the native PDAC TME. We show in preliminary data that Smoothened inhibition (SMOi) decreases proliferation and activity of myCAFs, but provokes the expansion of CD11b-positive myeloid cells in vivo. Thus, we hypothesize that LIS in PDAC is maintained by a delicate balance between myCAFs and myeloid cells, preventing effective T cell invasion. Single cell RNA-seq data comparing ctrl vs. SMOi-treated murine PDAC elucidates stromal subpopulations involved in the LIS phenotype and guides the identification of myeloid subtypes emerging after SMOi. Strikingly, we demonstrated that simultaneous SMOi and targeting myeloid cells via anti-Gr1 or CCR1 inhibition (CCR1i) significantly elevates cytotoxic T cell numbers within the TME. We are currently investigating whether the activity of these T cells may be further potentiated through combination with immunomodulatory agents. By testing various treatment combination in the same TME, we will identify the best synergistic effects for future immunotherapy approaches in human PDAC. In summary, we are elucidating the complex mechanism behind LIS in PDAC by employing our novel explant culture system alongside in vivo studies. We aim to develop a translatable regimen to neutralize LIS, reactivating the cytotoxic T cells in the tumor periphery to invade, proliferate, and attack cancer cells. Citation Format: Marie C. Hasselluhn, Lukas J. Vlahos, Dafydd Thomas, Alvaro Curiel Garcia, Amanda R. Decker, Tanner C. Dalton, Stephen A. Sastra, Carmine F. Palermo, Andrea Califano, Kenneth P. Olive. Combination CAF/myeloid targeting in PDAC [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C032.

TMIC-45. DISTRIBUTION AND TUMOR CELL PROXIMITY OF IMMUNE CELLS IN THE TUMOR CORE, INFILTRATION ZONE AND PERIPHERY OF GLIOBLASTOMAS

Abstract Glioblastoma is the most frequent and malignant brain tumor. Immune therapies have had limited effect, but not much is known about the frequency and type of immune cells in the infiltration zone and periphery. In these areas, tumor cells migrate into the brain parenchyma and prevent total tumor resection thereby leading to recurrence. This study aims to quantify the type and distribution of immune cells in glioblastomas. A cohort of 67 glioblastomas was established containing tissue from central tumor and/or infiltration zone and/or tumor periphery. Patients were included if most tumor cells were positive in immunohistochemical staining for P53. A multiplex staining was established, where tissue sections were stained with P53 (tumor cells), FOXP3 (FOXP3+ regulatory T cells), CD8 (CD8+ cytotoxic T cells) and IBA1 (microglia/macrophages). Cells were counted in tumor core, infiltration zone and periphery using Visiopharm software based on training of convolutional neural networks. Moreover, the number of tumor cells with immune cells in proximity (30 µm) was quantified. The number of CD8+, FOXP3+ and Iba1+ cells was significantly higher in the core than in the periphery (P &amp;lt; 0.001), and in the infiltration zone than in the periphery (P &amp;lt; 0.001). However, the CD8+, FOXP3+ and Iba1+ cells/tumor cell ratio increased from core to infiltration zone and to periphery (P &amp;lt; 0.001). There was a higher amount of tumor cells with CD8+ cells in proximity in the tumor core (P &amp;lt; 0.001), whereas the highest amount of tumor cells with IBA1+ cells and FOXP3+ cells in proximity was the infiltration zone (P &amp;lt; 0.001). In conclusion, although levels of immune cells are highest in the tumor core - the higher proximity and ratio of immune cells to tumor cells in the infiltration zone/periphery suggest immune-suppressive mechanisms to be active in these areas.