Elevated Interstitial Fluid Pressure Research Articles

Near-infrared Light-Triggered Size-Shrinkable theranostic nanomicelles for effective tumor targeting and regression.

Most nanomedicines with suitable sizes (normally 100-200 nm) exhibit favorable accumulation in the periphery of tumors but hardly penetrate into deep tumors. Effective penetration of nanomedicines requires smaller sizes (less than 30 nm) to overcome the elevated tumor interstitial fluid pressure. Moreover, integrating an efficient diagnostic agent in the nanomedicines is in high demand for precision theranostics of tumors. To this end, a near-infrared light (NIR) -triggered size-shrinkable micelle system (Fe3O4@AuNFs/DOX-M) coloaded antitumor drug doxorubicin (DOX) and biomodal imaging agent magnetic gold nanoflower (Fe3O4@AuNFs) was developed to achieve efficient theranostic of tumors. Upon the accumulation of Fe3O4@AuNFs/DOX-M in the tumor periphery, a NIR laser was irradiated near the tumor site, and the loaded Fe3O4@Au NFs could convert the light energy to heat, which triggered the cleavage of DOX-M to the ultra-small micelles (∼5 nm), thus realizing the deep penetration of micelles and on-demand drug release. Moreover, Fe3O4@AuNFs in the micelles could also be used as CT/MRI dual-modal contrast agent to "visualize" the tumor. Up to 92.6 % of tumor inhibition was achieved for the developed Fe3O4@AuNFs/DOX-M under NIR irradiation. This versatile micelle system provided a promising drug carrier platform realizing efficient tumor dual-modal diagnosis and photothermal-chemotherapy integration.

Mechanical regulation of mitochondrial morphodynamics in cancer cells by extracellular microenvironment

Recently, it has been recognized that physical abnormalities (e.g. elevated solid stress, elevated interstitial fluid pressure, increased stiffness) are associated with tumor progression and development. Additionally, these mechanical forces originating from tumor cell environment through mechanotransduction pathways can affect metabolism. On the other hand, mitochondria are well-known as bioenergetic, biosynthetic, and signaling organelles crucial for sensing stress and facilitating cellular adaptation to the environment and physical stimuli. Disruptions in mitochondrial dynamics and function have been found to play a role in the initiation and advancement of cancer. Consequently, it is logical to hypothesize that mitochondria dynamics subjected to physical cues may play a pivotal role in mediating tumorigenesis. Recently mitochondrial biogenesis and turnover, fission and fusion dynamics was linked to mechanotransduction in cancer. However, how cancer cell mechanics and mitochondria functions are connected, still remain poorly understood. Here, we discuss recent studies that link mechanical stimuli exerted by the tumor cell environment and mitochondria dynamics and functions. This interplay between mechanics and mitochondria functions may shed light on how mitochondria regulate tumorigenesis.

Effect of interstitial fluid pressure on shear wave elastography: an experimental and computational study

Objective. An elevated interstitial fluid pressure (IFP) can lead to strain-induced stiffening of poroelastic biological tissues. As shear wave elastography (SWE) measures functional tissue stiffness based on the propagation speed of acoustically induced shear waves, the shear wave velocity (SWV) can be used as an indirect measurement of the IFP. The underlying biomechanical principle for this stiffening behavior with pressurization is however not well understood, and we therefore studied how IFP affects SWV through SWE experiments and numerical modeling. Approach. For model set-up and verification, SWE experiments were performed while dynamically modulating IFP in a chicken breast. To identify the confounding factors of the SWV-IFP relationship, we manipulated the material model (linear poroelastic versus porohyperelastic), deformation assumptions (geometric linearity versus nonlinearity), and boundary conditions (constrained versus unconstrained) in a finite element model mimicking the SWE experiments. Main results. The experiments demonstrated a statistically significant positive correlation between the SWV and IFP. The model was able to reproduce a similar SWV-IFP relationship by considering an unconstrained porohyperelastic tissue. Material nonlinearity was identified as the primary factor contributing to this relationship, whereas geometric nonlinearity played a smaller role. The experiments also highlighted the importance of the dynamic nature of the pressurization procedure, as indicated by a different observed SWV-IFP for pressure buildup and relaxation, but its clinical relevance needs to be further investigated. Significance. The developed model provides an adaptable framework for SWE of poroelastic tissues and paves the way towards non-invasive measurements of IFP.

Modulation of the Tumor Stroma and Associated Novel Nanoparticle Strategies to Enhance Tumor Targeting

Growth of malignant cells in solid tumors induces changes to the tumor microenvironment (TME). These changes result in promotion of tumor growth, invasion, and metastasis, but also in tumor resistance to drugs and radiotherapy. The enhanced permeability and retention (EPR) effect in neo-angiogenic tumor tissue enables the transport of therapeutic molecules from the circulation into the tumor, but studies show that further diffusion of these agents is often not sufficient for efficient tumor eradication. Despite the hyperpermeable vasculature facilitating the delivery of drugs and tracers, the high density of stromal cells and matrix proteins, in combination with the elevated interstitial fluid pressure in the microenvironment of solid tumors, presents a barrier which limits the delivery of compounds to the core of the tumor. Reversing the cancer-cell-induced changes to the microenvironment as well as novel nanoparticle strategies to circumvent tumor-induced stromal changes have therefore been suggested as potential methods to improve the delivery of therapeutic molecules and drug efficacy. Strategies to modulate the TME, i.e., normalization of tumor vasculature and depletion of excessive stromal proteins and cells, show promising results in enhancing delivery of therapeutic compounds. Modulation of the TME may therefore enhance the efficacy of current cancer treatments and facilitate the development of novel treatment methods as an alternative for invasive resection procedures.

Abstract B054: Investigating ER stress responses in pancreatic ductal adenocarcinoma under lipid imbalance

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer-related death with a five-year survival rate of 10%. Dense desmoplasia results in poor intratumoral perfusion and elevated interstitial fluid pressure, creating regions with severe oxygen and nutrient deprivation. One result is a deficiency in unsaturated fatty acids (uFAs), as the de novo synthesis of uFAs requires oxygen. Our results indicate that PDAC cells experience endoplasmic reticulum (ER) stress and trigger subsequent ER stress responses under uFA limited conditions in vitro. ER stress responses are governed by three major stress sensors: Inositol-requiring enzyme 1𝛼 (IRE1𝛼), PKR-like ER kinase (PERK) and activating transcription factor 6 (ATF6). ER stress responses are also activated in PDAC murine and patient samples. Interestingly, the expression of ER stress markers is much higher in malignant lesions than benign pancreatic intraepithelial neoplasia. In contrast to tumor promoting, we found that PERK, ATF6 and IRE1𝛼 - XBP1 are cytotoxic to PDAC cells in vitro, as their knockout improved viability under stress conditions. Surprisingly, pharmacological inhibitor of XBP1 splicing, B-I09, didn’t recapitulate the results of the genetic approach. While B-I09 has cytoprotective effects in some PDAC lines, it is cytotoxic in others. In addition to inhibiting XBP1 splicing, B-I09 also inhibits regulated IRE1𝛼 dependent decay (RIDD). We speculate that RIDD might have opposite roles to XBP1s, leading to the discrepancy in the cellular phenotypes we observed. Most B-I09 responders are reported to be the basal-like transcriptional subtype. Therefore, we hypothesize that the basal-like subtype is more susceptible to B-I09. So far, we have shown that basal-like patient tumors are significantly enriched in the ER stress response pathways from bulk patient RNA-seq datasets, and we are currently validating this result in the publicly available scRNA-seq datasets from patients which contain basal-like and classical subtypes. We have generated isogenic models with basal-like features, such as epithelial to mesenchymal transition and deltaNP63 overexpression. However, our results suggest that these basal-like features did not sensitize classical PDAC cells to B-I09. Our lab has previously shown that B-I09 is selectively lethal to the MYC-transformed tumors. We are currently testing whether MYC overexpression would sensitize the non-responders to B-I09. It is exciting to see that some of the PDAC lines such as PANC1 and KPC murine line 4662 have reduced viability under B-I09 treatment. However, in our subcutaneous xenograft and allograft experiments, B-I09 only leads to a mild tumor growth suppression, which is unlikely to be therapeutically beneficial by itself. We are currently testing whether XBP1 inhibition could result in an enhanced tumor suppression using OVA antigen stimulated system, as XBP1 inhibition in tumor cells was shown to have tumor-repressing roles via immune system. Citation Format: Yanqing (Christine) Jiang, Xu Han, Nathan Coffey, Mai Wang, John Tobias, Raymond Ng, Carson Poltorack, M. Celeste Simon. Investigating ER stress responses in pancreatic ductal adenocarcinoma under lipid imbalance [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B054.

Hydraulic conductivity of human cancer tissue: A hybrid study.

Elevated tumor tissue interstitial fluid pressure (IFP) is an adverse biomechanical biomarker that predicts poor therapy response and an aggressive phenotype. Advances in functional imaging have opened the prospect of measuring IFP non-invasively. Image-based estimation of the IFP requires knowledge of the tissue hydraulic conductivity (K), a measure for the ease of bulk flow through the interstitium. However, data on the magnitude of K in human cancer tissue are not available. We measured the hydraulic conductivity of tumor tissue using modified Ussing chambers in surgical resection specimens. The effect of the tumor microenvironment (TME) on K was investigated by quantifying the collagen content, cell density, and fibroblast density of the tested samples using quantitative immune histochemistry. Also, we developed a computational fluid dynamics (CFD) model to evaluate the role of K on interstitial fluid flow and drug transport in solid tumors. The results show that the hydraulic conductivity of human tumor tissues is very limited, ranging from approximately 10-15 to 10-14 m2/Pa∙s. Moreover, K values varied significantly between tumor types and between different samples from the same tumor. A significant inverse correlation was found between collagen fiber density and hydraulic conductivity values. However, no correlation was detected between K and cancer cell or fibroblast densities. The computational model demonstrated the impact of K on the interstitial fluid flow and the drug concentration profile: higher K values led to a lower IFP and deeper drug penetration. Human tumor tissue is characterized by a very limited hydraulic conductivity, representing a barrier to effective drug transport. The results of this study can inform the development of realistic computational models, facilitate non-invasive IFP estimation, and contribute to stromal targeting anticancer therapies.

Overcoming tumor microenvironment obstacles: Current approaches for boosting nanodrug delivery.

In order to achieve targeted delivery of anticancer drugs, efficacy improvement, and side effect reduction, various types of nanoparticles are employed. However, their therapeutic effects are not ideal. This phenomenon is caused by tumor microenvironment abnormalities such as abnormal blood vessels, elevated interstitial fluid pressure, and dense extracellular matrix that affect nanoparticle penetration into the tumor's interstitium. Furthermore, nanoparticle properties including size, charge, and shape affect nanoparticle transport into tumors. This review comprehensively goes over the factors hindering nanoparticle penetration into tumors and describes methods for improving nanoparticle distribution by remodeling the tumor microenvironment and optimizing nanoparticle physicochemical properties. Finally, a critical analysis of future development of nanodrug delivery in oncology is further discussed. STATEMENT OF SIGNIFICANCE: This article reviews the factors that hinder the distribution of nanoparticles in tumors, and describes existing methods and approaches for improving the tumor accumulation from the aspects of remodeling the tumor microenvironment and optimizing the properties of nanoparticles. The description of the existing methods and approaches is followed by highlighting their advantages and disadvantages and put forward possible directions for the future researches. At last, the challenges of improving tumor accumulation in nanomedicines design were also discussed. This review will be of great interest to the broad readers who are committed to delivering nanomedicine for cancer treatment.

Abstract 594: Effects of interstitial pressure on lung cancer cells

Abstract Chronic inflammation is a risk factor for various types of cancer. The pressure in the interstitial tissues has been reported to be elevated in chronic inflammation and cancer. However, little is known about the biological effect of pressure from the basal side on epithelia, and a significance of the elevated interstitial fluid pressure in cancer biology is still poorly understood. In this study, we investigated the effect of hydrostatic pressure on cultured lung cancer cells grown on Transwell permeable filters by changing the height of the surfaces of culture medium in the apical and basal sides. A gradient of the hydrostatic pressure from the basal to the apical side induced stratification of epithelia. The elimination of the hydrostatic pressure gradient restored the stratification of epithelia. Interestingly, cavities were found within the stratified epithelia, and the characteristics of apical cell polarity including microvilli and tight junctions were formed in the surfaces the cavities. The cavities with similar aberrant cell polarity were also observed in human lung cancer tissues. The hydrostatic pressure gradient from the basal side also promoted cell proliferation and suppressed apoptosis. On the other hand, the expression levels of phosphorylated ERK and AKT were not significantly affected by the hydrostatic pressure. These results indicate that the pressure from the basal side to the epithelia promotes cell proliferation, suppresses apoptosis and induces aberrant cell polarity, which results in epithelial stratification. The findings in this study suggest that the elevated interstitial fluid pressure in chronic inflammation and cancer tissues facilitates cancer growth and plays a significant role in cancer biology. Citation Format: Shinsaku Tokuda, Keisuke Onoi, Tadaaki Yamada, Koichi Takayama. Effects of interstitial pressure on lung cancer cells [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 594.

Abstract 589: Long-term acclimation of target cell lines to tumor microenvironment culture condition provides mechanistic insights into cell therapy effectiveness

Abstract The tumor microenvironment (TME) has several characteristics that distinguish it from normal tissue, including elevated interstitial fluid pressures and hypoxia. To study the effects of TME culture conditions, commonly used target cell lines were serially passaged under hyperbaric and hypoxic conditions for a period of eight weeks. Conventionally expanded parental cell lines (normoxia) were compared against TME-acclimated tumor cell lines (TACTLs; hyperbaric and hypoxic), using RNAseq, ATACseq, and flow cytometry analysis. Commonly used target cell lines for cell therapy development were selected for TME acclimation (SKOV3, A549, JEKO1, and NALM6). TACTLs were generated using an AVATAR system, with culture conditions set at 1% O2 and 2.0 PSI, and serially passaged twice a week for a minimum of 8 weeks. Cell doubling times were measured weekly and compared against parental lines maintained under normoxic culture conditions in a conventional CO2 incubator. In-depth characterization was performed on TME-Acclimated Tumor Cell Lines (TACTLs), utilizing a multi-omic approach. Differential gene expression analysis was performed using RNAseq datasets, and surface biomarkers/targets used for cell therapy development were assessed via nanostring and flow cytometry. Growth kinetics and cell doubling times of TACTLs were initially inhibited during the first two weeks of culture under TME conditions, but eventually reached parity with their normoxia maintained parental cell lines at 6 to 8 weeks, signaling a successful adaptation of the tumor lines to low oxygen and hyperbaric conditions. RNAseq analysis revealed upregulation in glycolytic pathways and epithelial-to-mesenchymal signaling, accompanied by altered metabolic profiles. Surface target expression showed increased expression of checkpoint ligands, such as PD-L1. Cell therapy targets, such as ROR1 were also upregulated in A549 and JEKO1 cell lines passaged under TME. Preliminary drug screening experiments were conducted using checkpoint inhibitors and CAR-T candidates on TACTLs, revealing significant changes in half maximal inhibitory concentration (IC50) when compared to parental tumor lines maintained under normoxia. In summary, acclimation of tumor cell lines to TME culture conditions can provide unique mechanistic insights that can facilitate drug development efforts. Future studies will incorporate TACTLs for CDX tumor models with the goal of identifying cell therapies that work effectively in the tumor microenvironment. Citation Format: James Lim, Albert Wong, Ann Lu, Candy Garcia, Evan Massi, Yewei Xing, Ningchun Liu, Scott Wise. Long-term acclimation of target cell lines to tumor microenvironment culture condition provides mechanistic insights into cell therapy effectiveness [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 589.

Abstract B010: Investigating endoplasmic reticulum stress responses in pancreatic ductal adenocarcinoma under lipid imbalance

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer-related death with a five-year survival rate of 10%. The complex genetic makeup and heterogeneity of its microenvironment make PDAC difficult to treat. Desmoplasia, a major feature of PDAC’s TME, are dense fibrotic and inflammatory stromal regions. Desmoplastic reaction results in poor intratumoral perfusion and elevated interstitial fluid pressure, creating regions with severe oxygen and nutrient deprivation. One result is a deficiency in unsaturated fatty acids (uFAs) because the de novo synthesis of uFAs requires oxygen. Our results indicate that PDAC cells experience endoplasmic reticulum (ER) stress and trigger subsequent ER stress responses under uFA limited conditions in vitro. The ER stress responses are governed by three major stress sensors: Inositol-requiring enzyme 1𝛼 (IRE1𝛼), PKR-like ER kinase (PERK) and activating transcription factor 6𝛼 (ATF6𝛼). We hypothesize that ER stress responses are tumor promoting in PDAC and used as a survival strategy under uFA deprivation. In addition, previous studies showed that MYC-driven cancer cells are highly reliant on the ER stress response pathways. Therefore, we hypothesize that the two major PDAC transcriptional subtypes “Classical” and “Basal-like” have distinct dependencies on ER stress response pathways, with the basal-like subtype being more dependent due to an enrichment of MYC gene signatures. Our results suggest that opposite to cancer cell promoting, ER stress responses mediated by IRE1𝛼, PERK and ATF6𝛼 are cytotoxic in vitro under uFA deprived conditions. By analyzing the published bulk RNA-seq from patient samples, we found that the basal-like PDAC subtype is enriched with an ER stress gene signature. To avoid contamination from other cell populations, we are currently analyzing a published scRNA-seq dataset from 15 independent patient samples. We will investigate whether the ER stress signature is also enhanced in basal-like cells. Basal-like PDAC cells are usually more mesenchymal; we therefore created isogenic “epithelial” and “mesenchymal” lines to mimic PDAC subtypes. However, we did not see signaling preferences between the lines. One caveat is that the basal-like subtype have many more features than being mesenchymal, and our isogenic system might not be a faithful modeling of the subtypes. We are studying whether hypoxic PDAC cells experience ER stress in vivo due to a deprivation in uFAs using multiplex fluorescent imaging, stimulated Raman microscopy and/or mass spectrometry. We are evaluating the role of ER stress responses in vivo and see whether inhibiting ER stress response alone or in combination with other reagents gives rise to potential therapeutic opportunities. This project will provide a better understanding of the role ER stress responses play under uFA deprivation and will also enable us to further understand PDAC subtypes in terms of how they utilize ER stress responses. Citation Format: Yanqing (Christine) Jiang, Xu Han, Mai Wang, Raymond Ng, Pamela Burgess-Jones, John Tobias, Nathan Coffey, Andrew Hu, Brian Keith, Celeste Simon. Investigating endoplasmic reticulum stress responses in pancreatic ductal adenocarcinoma under lipid imbalance [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 B010.

Abstract 3809: Adaptation of prostate cancer cell lines to tumor microenvironment culture conditions induces expression of androgen receptor splice variant 7 and contributes to drug resistance

Abstract The tumor microenvironment (TME) has several characteristics that distinguish it from normal tissue, including elevated interstitial fluid pressures and hypoxic environments. Both pressurized and hypoxic culture conditions can influence gene and protein expression of tumor cells which can result in tumor metastasis and drug resistance. Enzalutamide is effective in treating metastatic castration-resistant prostate cancer (mCRPC), but majority of patients eventually develop resistance to enzalutamide. One of the biomarkers of enzalutamide resistance is the expression of androgen receptor splice variant 7 (AR-V7), but the mechanism of how AR-V7 expression is upregulated in prostate cancer is unclear. To investigate the influence of TME culture conditions on prostate cancer cell lines, 22Rv1 and LNCaP cell lines were incubated under pressurized and hypoxic culture conditions (2 PSI + 1%O2) for a minimum of 7 days. We compared cell growth, morphology changes, gene expression profiles and drug response to enzalutamide treatment. The growth of 22Rv1 and LNCaP cells was markedly inhibited under TME culture conditions, accompanied by distinct changes in cell morphology. 22Rv1 and LNCaP cells cultured under TME conditions exhibited increased resistance to enzalutamide treatment. Higher levels of AR-V7 expression were detected in 22Rv1 and LNCaP cell lines maintained for 7 days in TME culture conditions. RNA-seq data identified 700+ genes that were differentially expressed in 22Rv1 and LNCaP cell lines adapted to TME culture conditions. GSEA analysis identified signaling pathways associated with hypoxia, glycolysis and epithelial-mesenchymal transition were upregulated under TME culture conditions, and the pathways of xenobiotic metabolism and oxidative phosphorylation were downregulated by TME culture conditions. In summary, adaptation to TME culture conditions can promote the expression of AR-V7 in prostate cancer cell lines and can contribute to enzalutamide drug resistance. Adaptation of cell lines to TME culture conditions can provide meaningful insights into mechanisms of drug resistance while providing a novel approach to identify targets and biomarkers of drug resistance. Citation Format: Yunmin Li, Albert Wong, Ann Lu, James Lim. Adaptation of prostate cancer cell lines to tumor microenvironment culture conditions induces expression of androgen receptor splice variant 7 and contributes to drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3809.

The Hippo pathway drives the cellular response to hydrostatic pressure.

Cells need to rapidly and precisely react to multiple mechanical and chemical stimuli in order to ensure precise context‐dependent responses. This requires dynamic cellular signalling events that ensure homeostasis and plasticity when needed. A less well‐understood process is cellular response to elevated interstitial fluid pressure, where the cell senses and responds to changes in extracellular hydrostatic pressure. Here, using quantitative label‐free digital holographic imaging, combined with genome editing, biochemical assays and confocal imaging, we analyse the temporal cellular response to hydrostatic pressure. Upon elevated cyclic hydrostatic pressure, the cell responds by rapid, dramatic and reversible changes in cellular volume. We show that YAP and TAZ, the co‐transcriptional regulators of the Hippo signalling pathway, control cell volume and that cells without YAP and TAZ have lower plasma membrane tension. We present direct evidence that YAP/TAZ drive the cellular response to hydrostatic pressure, a process that is at least partly mediated via clathrin‐dependent endocytosis. Additionally, upon elevated oscillating hydrostatic pressure, YAP/TAZ are activated and induce TEAD‐mediated transcription and expression of cellular components involved in dynamic regulation of cell volume and extracellular matrix. This cellular response confers a feedback loop that allows the cell to robustly respond to changes in interstitial fluid pressure.

Multistage pH-responsive codelivery liposomal platform for synergistic cancer therapy

BackgroundSmall interfering RNA (siRNA) is utilized as a potent agent for cancer therapy through regulating the expression of genes associated with tumors. While the widely application of siRNAs in cancer treatment is severely limited by their insufficient biological stability and its poor ability to penetrate cell membranes. Targeted delivery systems hold great promise to selectively deliver loaded drug to tumor site and reduce toxic side effect. However, the elevated tumor interstitial fluid pressure and efficient cytoplasmic release are still two significant obstacles to siRNA delivery. Co-delivery of chemotherapeutic drugs and siRNA represents a potential strategy which may achieve synergistic anticancer effect. Herein, we designed and synthesized a dual pH-responsive peptide (DPRP), which includes three units, a cell-penetrating domain (polyarginine), a polyanionic shielding domain (ehG)n, and an imine linkage between them. Based on the DPRP surface modification, we developed a pH-responsive liposomal system for co-delivering polo-like kinase-1 (PLK-1) specific siRNA and anticancer agent docetaxel (DTX), D-Lsi/DTX, to synergistically exhibit anti-tumor effect.ResultsIn contrast to the results at the physiological pH (7.4), D-Lsi/DTX lead to the enhanced penetration into tumor spheroid, the facilitated cellular uptake, the promoted escape from endosomes/lysosomes, the improved distribution into cytoplasm, and the increased cellular apoptosis under mildly acidic condition (pH 6.5). Moreover, both in vitro and in vivo study indicated that D-Lsi/DTX had a therapeutic advantage over other control liposomes. We provided clear evidence that liposomal system co-delivering siPLK-1 and DTX could significantly downregulate expression of PLK-1 and inhibit tumor growth without detectable toxic side effect, compared with siPLK-1-loaded liposomes, DTX-loaded liposomes, and the combinatorial administration.ConclusionThese results demonstrate great potential of the combined chemo/gene therapy based on the multistage pH-responsive codelivery liposomal platform for synergistic tumor treatment.Graphical

Antigen Receptor T Cells (CAR-T) Effectively Control Tumor Growth in a Colorectal Liver Metastasis Model.

Effective treatment of solid tumors requires multi-modality approaches. In many patients with stage IV liver disease, current treatments are not curative. Chimeric antigen receptor T cells (CAR-T) are an intriguing option following success in hematological malignancies, but this has not been translated to solid tumors. Limitations include sub-optimal delivery and elevated interstitial fluid pressures. We developed a murine model to test the impact of high-pressure regional delivery (HPRD) on trafficking to liver metastases (LM) and tumor response. CAR-T were generated from CD45.1 mice and adoptively transferred into LM-bearing CD45.2 mice via regional or systemic delivery (RD, SD). Trafficking, tumor growth, and toxicity were evaluated with flow cytometry, tumor bioluminescence (TB, photons/sec log2-foldover baseline), and liver function tests (LFTs). RD of CAR-T was more effective at controlling tumor growth versus SD from post-treatment days (PTD) 2-7 (P = 0.002). HPRD resulted in increased CAR-T penetration versus low-pressure RD (LPRD, P = 0.004), suppression of tumor proliferation (P = 0.03), and trended toward improved long-term control at PTD17 (TB=3.7 versus 6.1, P = 0.47). No LFT increase was noted utilizing HPRD versus LPRD (AST/ALT P = 0.65/0.84) while improved LFTs in RD versus SD groups suggested better tumor control (HPRD AST/ALT P = 0.04/0.04, LPRD AST/ALT P = 0.02/0.02). Cellular immunotherapy is an emerging option for solid tumors. Our model suggests RD and HPRD improved CAR-T penetration into solid tumors with improved short-term tumor control. Barriers associated with SD can be overcome using RD techniques to maximize therapeutic delivery and HPRD may further augment efficacy without increased toxicity.

Regulation of Tumor Invasion by the Physical Microenvironment: Lessons from Breast and Brain Cancer.

The success of anticancer therapies is often limited by heterogeneity within and between tumors. While much attention has been devoted to understanding the intrinsic molecular diversity of tumor cells, the surrounding tissue microenvironment is also highly complex and coevolves with tumor cells to drive clinical outcomes. Here, we propose that diverse types of solid tumors share common physical motifs that change in time and space, serving as universal regulators of malignancy. We use breast cancer and glioblastoma as instructive examples and highlight how invasion in both diseases is driven by the appropriation of structural guidance cues, contact-dependent heterotypic interactions with stromal cells, and elevated interstitial fluid pressure and flow. We discuss how engineering strategies show increasing value for measuring and modeling these physical propertiesfor mechanistic studies. Moreover, engineered systems offer great promise for developing and testing novel therapies that improve patient prognosis by normalizing the physical tumor microenvironment.

Interstitial Fluid Pressure and Hydraulic Conductivity in Solid Tumors: Introducing an In-Vitro Method for Measuring the Hydraulic Conductivity of Tumor Tissue

Background: Resistance against drug delivery is a challenge in oncology, preventing the development of optimal therapeutic outcomes. This has been related to a group of hostile biophysical characteristics in the tumor microenvironment. Among them, the elevated interstitial fluid pressure (IFP) in solid tumors has been identified as a barrier for drug transport. Also, the hydraulic conductivity (K) of tumor stroma strongly correlates with the IFP. However, values of K in human cancer tissues are quite limited.

Abstract PO-027: Investigating unfolded protein responses in pancreatic ductal adenocarcinoma under lipid imbalance

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer-related death with a five-year survival rate of only 10%. A major feature of PDAC tumor microenvironment (TME) is desmoplasia, which are dense fibrotic and inflammatory stromal regions constituting the PDAC TME. This desmoplastic reaction results in poor intratumoral perfusion and elevated interstitial fluid pressure, creating regions with severe oxygen and nutrient deprivation. One result is a significant deficiency in unsaturated fatty acids (UFAs) because the de novo synthesis of UFAs requires molecular oxygen. Our data indicate that PDAC cells experience endoplasmic reticulum (ER) stress and trigger a subsequent unfolded protein response (UPR) under tumor-like stress, which can be mitigated by UFAs. The UPR regulates an intricate signaling network mediated by three major stress sensors: Inositol-requiring enzyme 1a (IRE1a), PKR-like ER kinase (PERK) and activating transcription factor 6a (ATF6a). UPR output can be either cytoprotective or cytotoxic depending on tumor types and the severity of stress. Our data suggest that B-I09, a selective IRE1 RNase inhibitor, impairs PDAC cell viability under stress conditions, indicating a pro-survival role for IRE1a. Previous studies showed that MYC-driven cancer cells are highly reliant on UPR pathways. Therefore, we hypothesize that the two major PDAC transcriptional subtypes “Classical” and “Basal-like” may have distinct dependencies on UPR pathways, with the Basal-like subtype being more dependent due to an enrichment of MYC gene signatures. Overall, we hypothesize that IRE1a, PERK and/or ATF6a play(s) an oncogenic role in PDAC tumorigenesis due to lipid saturation abnormalities, and the Basal-like PDAC subtype is more sensitive to inhibition of UPR pathway(s). We are evaluating the role of IRE1a, PERK and ATF6a using pharmacologic inhibition and genetic knockdown in vitro and in vivo under UFA deprived conditions. We will also evaluate PDAC intracellular and interstitial fluid lipidomics using LC-MS. In addition, we will assess whether hypoxic PDAC cells experience ER stress in vivo due to UFA deprivation using Stimulated Raman scattering (SRS) microscopy. To explore the subtype-specific dependency on the UPR pathways, we are querying a UPR gene signature in different tumor cell clusters using single cell RNA-seq datasets of patient samples. In addition, we will employ an isogenic system for PDAC subtypes and examine subtype-specific responses to UPR inhibition in vitro and in vivo. Our goal is to better understand UPR pathways in PDAC cells as a survival mechanism in response to the “lipotoxicity” induced by ER membrane saturation, with a consideration of intratumoral heterogeneity of PDAC cell subtype. Citation Format: Yanqing (Christine) Jiang, Xu Han, Michelle Burrows, Carson Poltorack, Celeste Simon, Brian Keith. Investigating unfolded protein responses in pancreatic ductal adenocarcinoma under lipid imbalance [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-027.

The Role of Ultrasound in Modulating Interstitial Fluid Pressure in Solid Tumors for Improved Drug Delivery.

The unique microenvironment of solid tumors, including desmoplasia within the extracellular matrix, enhanced vascular permeability, and poor lymphatic drainage, leads to an elevated interstitial fluid pressure which is a major barrier to drug delivery. Reducing tumor interstitial fluid pressure is one proposed method of increasing drug delivery to the tumor. The goal of this topical review is to describe recent work using focused ultrasound with or without microbubbles to modulate tumor interstitial fluid pressure, through either thermal or mechanical effects on the extracellular matrix and the vasculature. Furthermore, we provide a review on techniques in which ultrasound imaging may be used to diagnose elevated interstitial fluid pressure within solid tumors. Ultrasound-based techniques show high promise in diagnosing and treating elevated interstitial pressure to enhance drug delivery.

A computational model of glioma reveals opposing, stiffness-sensitive effects of leaky vasculature and tumor growth on tissue mechanical stress and porosity.

A biphasic computational model of a growing, vascularized glioma within brain tissue was developed to account for unique features of gliomas, including soft surrounding brain tissue, their low stiffness relative to brain tissue, and a lack of draining lymphatics. This model is the first to couple nonlinear tissue deformation with porosity and tissue hydraulic conductivity to study the mechanical interaction of leaky vasculature and solid growth in an embedded glioma. The present model showed that leaky vasculature and elevated interstitial fluid pressure produce tensile stress within the tumor in opposition to the compressive stress produced by tumor growth. This tensile effect was more pronounced in softer tissue and resulted in a compressive stress concentration at the tumor rim that increased when tumor was softer than host. Aside from generating solid stress, fluid pressure-driven tissue deformation decreased the effective stiffness of the tumor while growth increased it, potentially leading to elevated stiffness in the tumor rim. A novel prediction of reduced porosity at the tumor rim was corroborated by direct comparison with estimates from our in vivo imaging studies. Antiangiogenic and radiation therapy were simulated by varying vascular leakiness and tissue hydraulic conductivity. These led to greater solid compression and interstitial pressure in the tumor, respectively, the former of which may promote tumor infiltration of the host. Our findings suggest that vascular leakiness has an important influence on in vivo solid stress, stiffness, and porosity fields in gliomas given their unique mechanical microenvironment.

In silico investigations of intratumoral heterogeneous interstitial fluid pressure

Recent preclinical studies have shown that interstitial fluid pressure (IFP) within tumors can be heterogeneous Andersen et al. (2019). In that study tumors of two xenograft models, respectively, HL-16 cervical carcinoma and Panc-1 pancreatic carcinoma, were investigated. Significant heterogeneity in IFP was reported and it was proposed that this was associated with division of tissue into compartments separated by thick connective tissue bands for the HL-16 tumors and with dense collagen-rich extracellular matrix for the Panc-1 tumors. The purpose of the current work is to explore these experimental observations by using in silico generated tumor models. We consider a mathematical multiphase model which accounts for tumor cells, fibroblasts and interstitial fluid. The model has been trained to comply with experimental in vitro results reported in Shieh et al. (2011) which has identified autologous chemotaxis, ECM remodeling, and cell-fibroblast interaction as drivers for invasive tumor cell behavior. The in silico model is informed with parameters that characterize the leaky intratumoral vascular network, the peritumoral lymphatics which collect the fluid, and the density of ECM as represented through the hydraulic conductivity of the interstitial space. Heterogeneous distribution of solid stress may result in heterogeneous compression of blood vessels and, thus, heterogeneous vascular density inside the tumor. To mimic this we expose the in silico tumor to an intratumoral vasculature whose net effect of density of blood vesssels and vessel wall conductivity is varied through a 2D Gaussian variogram constrained such that the resulting IFPs lie within the range as reported from the preclinical study. The in silico cervical carcinoma model illustrates that sparse ECM was associated with uniform intratumoral IFP in spite of heterogeneous microvascular network, whereas compartment structures resulted in more heterogeneous IFP. Similarly, the in silico pancreatic model shows that heterogeneity in the microvascular network combined with dense ECM structure prevents IFP to even out and gives rise to heterogeneous IFP. The computer model illustrates how a heterogeneous invasive front might form where groups of tumor cells detach from the primary tumor and form isolated islands, a behavior which is natural to associate with metastatic propensity. However, unlike experimental studies, the current version of the in silico model does not show an association between metastatic propensity and elevated IFP.