Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer characterized by activating KRAS mutations and TP53 alterations. TP53 missense mutations lose their wild-type tumor-suppressor function. Here, we studied whether p53 missense mutations have potential gain-of-function oncogenic roles and their impact on cancer-cell-intrinsic gene expression and the tumor immune microenvironment (TME) in PDAC. p53R172H established an immunosuppressive TME and impaired the efficacy of immune checkpoint inhibitors (ICIs) by regulating a distinct set of chemokines. Among these, tumor-specific reduction of Cxcl1, which encodes a chemoattractant for neutrophils, promoted T cell infiltration and decreased tumor growth. Mechanistically, p53R172H occupied the distal enhancers of Cxcl1 and amplified its expression. These enhancers were responsible for Cxcl1 expression and were essential for its immunosuppressive function. Nuclear factor κB (NF-κB) was a critical cofactor required for p53R172H occupancy at these enhancers. Thus, a common mutation in a tumor-suppressor transcription factor appropriates enhancers, thereby stimulating chemokine expression and establishing an immunosuppressive TME that diminishes ICI efficacy in PDAC.

Prime editors make programmed genome modifications by writing new sequences into extensions of nicked DNA 3' ends1. These edited 3' new strands must displace competing 5' strands to install edits, yet a bias towards retaining the competing 5' strands hinders efficiency and can cause indel errors2. Here we discover that nicked end degradation, consistent with competing 5' strand destabilization, can be promoted by Cas9-nickase mutations that relax nick positioning. We exploit this mechanism to engineer efficient prime editors with strikingly low indel errors. Combining this error-suppressing strategy with the latest efficiency-boosting architecture, we design a next-generation prime editor (vPE). Compared with previous editors, vPE features comparable efficiency yet up to 60-fold lower indel errors, enabling edit:indel ratios as high as 543:1.

Abstract Pancreatic ductal Adenocarcinoma (PDAC) is an aggressive malignancy complicated by poor early diagnosis and a lack of response to traditional treatments. It is characterized by a desmoplastic stroma, a lack of infiltration and activation of T cells, and a low mutational burden. The genetic landscape of PDAC is defined by activating KRAS mutations (~90%) and p53 alterations (~70%), but the molecular switches perturbed by these genetic aberrations remain unclear. p53 missense mutations, unlike mutations resulting in the loss of p53, are considered to acquire tumor-supporting functions. But these novel functions remain uncharacterized. The ambiguity in the molecular mechanism of mutant-p53 is further exacerbated by the existence of various types of p53 mutations. The majority of p53 mutations are missense mutations in the DNA binding domain. The repertoire of transcription factors (TFs) it can interact with and the vast regulatory landscape of each TF—composed of gene promoters and distal enhancers—present obstacles in understanding the molecular mechanisms promoting PDAC. In this study, we examined how a common p53 missense mutation in PDAC plays a role in weakening the Immune checkpoint Inhibitors (ICIs) efficacy. Using cells derived from a genetically engineered mouse model (GEMM) of PDAC with activating KRAS mutation (KrasG12D/+) and a p53 missense mutation (p53R172H/-), we found that the PDAC tumorigenesis and resistance to ICIs are dependent on the mutant-p53. We used isogenic p53-null PDAC cells and the restoration of p53R172H in p53-null cells to demonstrate the role of p53R172H in controlling the expression of immunosuppressive chemokine genes such as Cxcl1. p53R172H deletion attenuated PDAC tumor growth, increased the influx of cytotoxic T-cells, and sensitized the tumor to ICIs. The p53R172H-mediated TME reprogramming was replicated by the deletion of the Cxcl1 gene, suggesting the anti-tumorigenic effect of p53R172H was mediated by the Cxcl1 gene. We probed the mechanism of Cxcl1 expression dependence on p53R172H. We found that in conjunction with NF-kB, p53R172H occupies the distal transcription regulatory elements (dTREs) of the Cxcl1 gene harboring NF-kB binding sites. Strikingly, deletion of the Cxcl1 dTREs in PDAC cells recapitulates the phenotypes of p53R172H deletion and Cxcl1 deletion in terms of tumor size, immune landscape of the TME, and ICI responsiveness. Furthermore, we examined the interplay between p53R172H and NF-kB and found that the p53R172H physically interacts with the NF-kB subunit RelA and facilitates its nuclear translocation. Overall, we characterize how a common p53 mutation in PDAC co-opts non-coding regulatory DNA to augment the expression of selective chemokine genes and establishes an immunosuppressive TME to shield the therapeutic benefits of ICIs. Citation Format: Dig B. Mahat, Heena Kumra, Emily Metcalf, Sarah Castro, Kim Nguyen1, Arundeep Singh, William W. Ho, Ivy Chen, Brandon Sullivan, Leon Yim, Enrico Moiso, Vikash Chauhan, Hernandez Moura Silva, Stefani Spranger, Rakesh Jain, Phillip A. Sharp. Mutant-p53 amplifies Cxcl1 expression from distal enhancers blunting immune checkpoint inhibition efficacy in pancreatic cancer [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 B087.

Background Myeloid cells, unlike other immune cells such as T cells or NK cells, are known residents in the solid tumor microenvironment (TME). In the absence of checkpoints and in proinflammatory conditions, M1 macrophages are known to be capable of direct phagocytosis of tumor cells and can present tumor-associated antigens to the host immune system. However, the immunosuppressive conditions within the TME restrict and limit the anti-tumor response of tumor associated macrophages (TAMs), including their ability to recruit and activate other immune cells against the tumor. Engineered CAR-Monocytes can serve a unique function in cell therapy by bridging a key gap in the treatment of solid tumors. We are developing an autologous engineered CAR-M cell therapy product targeting Glypican-3 to treat hepatocellular carcinoma. The CAR serves as a homing ‘GPS’ signal for trafficking directly to the tumor site, and directs phagocytosis specifically at targeted tumor cells. Our proprietary M83.CAR molecule contains a macrophage-specific costimulatory domain that significantly increases the phagocytosis function of the CAR-M cells. Furthermore, we have demonstrated that the M83.CAR-M cells are not inhibited by the prevalent CD47 ‘do not eat me’ checkpoint, which is known to restrict myeloid function in the TME. Methods Primary hematopoietic stem cells (HSCs) were harvested and engineered to express a CAR molecule by lentivirus transduction generating CAR-HSCs. The CAR-HSCs underwent our proprietary ex-vivo HSC differentiation process to yield CAR-Monocytes. Results Effective phagocytosis of engineered CAR-M cells is central to subsequent mechanisms of actions that can elicit robust anti-tumor immunity against patient-specific neoantigens. However, the first barrier to overcome is effective infiltration of engineered CAR-M into the tumor from the periphery. We have demonstrated that our engineered CAR-Monocyte drug product can successfully home to the targeted tumor specifically, from the periphery. Subsequent in situ differentiation of CAR-monocytes into CAR-macrophages enables robust tumor cell phagocytosis, the central mechanism that results in the following: 1) proinflammatory shift in the TME, 2) recruitment of APCs and immune cells, 3) activation of T-cells against tumor neoantigens. Conclusions The above summarizes a unique outcome of the CAR-M mechanism of action that is not capitulated by CAR-T or CAR-NK cells. Leveraging and further enhancing CAR-M function could lead to the rejection of the tumor and its metastases, particularly in combination with other immune-modulating therapies. Our proprietary HSC-derived CAR-M platform yields a unique product that demonstrates durability and superior function, which we expect to translate to the clinic.

To have the desired therapeutic effect, nanomedicines and macromolecular medications must move from the site of injection to the site of action, without having adverse effects. Transvascular transport is a critical step of this navigation, as exemplified by the Enhanced Permeability and Retention (EPR) effect in solid tumors, not found in normal organs. Numerous studies have concluded that passive, diffusion- and convection-based transport predominates over active, cellular mechanisms in this effect. However, recent work using a new approach reevaluated this principle by comparing tumors with or without fixation and concluded the opposite. Here, we address the controversy generated by this new approach by reporting evidence from experimental investigations and computer simulations that separate the contributions of active and passive transport. Our findings indicate that tissue fixation reduces passive transport as well as active transport, indicating the need for new methods to distinguish the relative contributions of passive and active transport.

CRISPR-Cas9 introduces targeted DNA breaks that engage competing DNA repair pathways, producing a spectrum of imprecise insertion/deletion mutations (indels) and precise templated mutations (precise edits). The relative frequencies of these pathways are thought to primarily depend on genomic sequence and cell state contexts, limiting control over mutational outcomes. Here, we report that engineered Cas9 nucleases that create different DNA break structures engage competing repair pathways at dramatically altered frequencies. We accordingly designed a Cas9 variant (vCas9) that produces breaks which suppress otherwise dominant nonhomologous end-joining (NHEJ) repair. Instead, breaks created by vCas9 are predominantly repaired by pathways utilizing homologous sequences, specifically microhomology-mediated end-joining (MMEJ) and homology-directed repair (HDR). Consequently, vCas9 enables efficient precise editing through HDR or MMEJ while suppressing indels caused by NHEJ in dividing and nondividing cells. These findings establish a paradigm of targeted nucleases custom-designed for specific mutational applications.

Supplementary Data from Multiphoton Phosphorescence Quenching Microscopy Reveals Kinetics of Tumor Oxygenation during Antiangiogenesis and Angiotensin Signaling Inhibition

Supplementary Tables S1 - S6. Supplementary Table S1. CT values for demosplasia-related genes in PAN02 tumors. Data obtained from PCR array. Supplementary Table S2. CT values for demosplasia-related genes in AK4.4 tumors. Data obtained from PCR array. Supplementary Table S3. Univariate analysis of prognostic factors for resected pancreatic cancer patients with body mass index (BMI) {less than or equal to}25. Supplementary Table S4. Multivariate analysis of prognostic factors for resected pancreatic cancer patients with body mass index (BMI) {less than or equal to}25. Supplementary Table S5. Univariate analysis of prognostic factors for resected pancreatic cancer patients with body mass index (BMI) >25. Supplementary Table S6. Multivariate analysis of prognostic factors for resected pancreatic cancer patients with body mass index (BMI) >25.

Liver metastasis is a major cause of mortality for patients with colorectal cancer (CRC). Mismatch repair-proficient (pMMR) CRCs make up about 95% of metastatic CRCs, and are unresponsive to immune checkpoint blockade (ICB) therapy. Here we show that mouse models of orthotopic pMMR CRC liver metastasis accurately recapitulate the inefficacy of ICB therapy in patients, whereas the same pMMR CRC tumors are sensitive to ICB therapy when grown subcutaneously. To reveal local, nonmalignant components that determine CRC sensitivity to treatment, we compared the microenvironments of pMMR CRC cells grown as liver metastases and subcutaneous tumors. We found a paucity of both activated T cells and dendritic cells in ICB-treated orthotopic liver metastases, when compared with their subcutaneous tumor counterparts. Furthermore, treatment with Feline McDonough sarcoma (FMS)-like tyrosine kinase 3 ligand (FIt3L) plus ICB therapy increased dendritic cell infiltration into pMMR CRC liver metastases and improved mouse survival. Lastly, we show that human CRC liver metastases and microsatellite stable (MSS) primary CRC have a similar paucity of T cells and dendritic cells. These studies indicate that orthotopic tumor models, but not subcutaneous models, should be used to guide human clinical trials. Our findings also posit dendritic cells as antitumor components that can increase the efficacy of immuno-therapies against pMMR CRC.

Significance Immune checkpoint blockade (ICB) has been efficacious in several cancer types. However, mismatch repair–proficient (pMMR) metastatic colorectal cancer (CRC), ∼95% of total metastatic CRC cases, typically does not respond to ICB. Here, we show that orthotopic liver metastasis mouse models of pMMR CRC cell lines are unresponsive to ICB and recapitulate the resistance of human disease, unlike subcutaneous tumors of the same cell lines. We also show that just like the human disease, orthotopic pMMR CRC liver metastases have a paucity of T cells and dendritic cells, and that treatment with Flt3L sensitizes the liver metastases to ICB. Our findings highlight that orthotopic tumor models, and not subcutaneous models, should be used for preclinical studies of cancer immunotherapy.

Genomic studies have significantly improved our understanding of hepatocellular carcinoma (HCC) biology and have led to the discovery of multiple protein-coding genes driving hepatocarcinogenesis. In addition, these studies have identified thousands of new non-coding transcripts deregulated in HCC. We hypothesize that some of these transcripts may be involved in disease progression. Long non-coding RNAs are a large class of non-coding transcripts which participate in the regulation of virtually all cellular functions. However, a majority of lncRNAs remain dramatically understudied. Here, we applied a pooled shRNA-based screen to identify lncRNAs essential for HCC cell survival. We validated our screening results using RNAi, CRISPRi, and antisense oligonucleotides. We found a lncRNA, termed ASTILCS, that is critical for HCC cell growth and is overexpressed in tumors from HCC patients. We demonstrated that HCC cell death upon ASTILCS knockdown is associated with apoptosis induction and downregulation of a neighboring gene, protein tyrosine kinase 2 (PTK2), a crucial protein for HCC cell survival. Taken together, our study describes a new, non-coding RNA regulator of HCC.

Cancer-associated fibroblasts (CAFs) can either suppress or support T lymphocyte activity, suggesting that CAFs may be reprogrammable to an immunosupportive state. Angiotensin receptor blockers (ARBs) convert myofibroblast CAFs to a quiescent state, but whether ARBs can reprogram CAFs to promote T lymphocyte activity and enhance immunotherapy is unknown. Moreover, ARB doses are limited by systemic adverse effects such as hypotension due to the importance of angiotensin signaling outside tumors. To enhance the efficacy and specificity of ARBs in cancer with the goal of revealing their effects on antitumor immunity, we developed ARB nanoconjugates that preferentially accumulate and act in tumors. We created a diverse library of hundreds of acid-degradable polymers and chemically linked ARBs to the polymer most sensitive to tumor pH. These tumor microenvironment-activated ARBs (TMA-ARBs) remain intact and inactive in circulation while achieving high concentrations in tumors, wherein they break down to active ARBs. This tumor-preferential activity enhances the CAF-reprogramming effects of ARBs while eliminating blood pressure-lowering effects. Notably, TMA-ARBs alleviate immunosuppression and improve T lymphocyte activity, enabling dramatically improved responses to immune-checkpoint blockers in mice with primary as well as metastatic breast cancer.

Significance Although immune checkpoint blockade (ICB) along with nab-paclitaxel has increased progression-free survival in triple-negative breast cancer patients, a large fraction of metastatic breast cancer (mBC) patients do not benefit from ICBs. The presence of a fibrotic tumor microenvironment can suppress the immune response to cancer. Here we found fibrosis and immunosuppression in both primary and metastatic breast cancer lesions. We show that targeting CXCR4/CXCL12 signaling, using plerixafor, an Food and Drug Administration-approved drug, reduces fibrosis, alleviates immunosuppression, and significantly enhances the efficacy of immune checkpoint blockers in preclinical models of mBC. Our findings provide a deeper understanding of mechanisms by which desmoplasia promotes immunosuppression in mBC and suggest a clinically translatable approach that can be combined with immunotherapy in patients to enhance therapeutic response.

Metastatic breast cancers (mBCs) are largely resistant to immune checkpoint blockade, but the mechanisms remain unclear. Primary breast cancers are characterized by a dense fibrotic stroma, which is considered immunosuppressive in multiple malignancies, but the stromal composition of breast cancer metastases and its role in immunosuppression are largely unknown. Here we show that liver and lung metastases of human breast cancers tend to be highly fibrotic, and unlike primary breast tumors, they exclude cytotoxic T-lymphocytes (CTLs). Unbiased analysis of the TCGA database of human breast tumors revealed a set of genes that are associated with stromal T-lymphocyte exclusion. Among these we focused on CXCL12 as a relevant target based on its known roles in immunosuppression in other cancer types. We found that the CXCL12 receptor CXCR4 is highly expressed in both human primary tumors and metastases. To gain insight into the role of CXCL12/CXCR4 axis, we inhibited CXCR4 and found that CXCR4 blockade decreases fibrosis, alleviates solid stress, decompresses blood vessels, increases CTL infiltration, and decreases immunosuppression in murine mBC models. We confirmed that these immunosuppressive effects are dependent on cancer-associated fibroblast (CAF) signaling through genetic deletion of Cxcr4 in aSMA+ cells. Accordingly, CXCR4 inhibition more than doubles the response to immune checkpoint blockers in mice bearing mBCs. These findings demonstrate that CXCL12/CXCR4-mediated desmoplasia in mBC promotes immunosuppression and is a potential target for overcoming therapeutic resistance to immune checkpoint blockade in mBC patients. Citation Format: Ivy X. Chen, Vikash P. Chauhan, Jessica Posada, Mei R. Ng, Michelle Wu, Pichet Adstamongkonkul, Peigen Huang, Neal Lindeman, Robert Langer, Rakesh K. Jain. Blocking CXCR4 alleviates desmoplasia, increases T-lymphocyte infiltration, and improves immunotherapy in metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2744.

Introduction: Increased tissue stiffness is a widely accepted and actively studied biomechanical property of desmoplastic tumors, and has been linked to several hallmarks of cancer, such as growth, invasion and metastasis. The abnormal mechanics of tumors, however, are not limited to tissue stiffening. We recently demonstrated that solid stress represents a new mechanopathology that is consistently elevated in mouse and human tumors. The solid stress, transmitted by solid elements of the extracellular matrix, is distinct from interstitial fluid pressure. Therefore, tumors are not only more rigid than many normal tissues, but cancer cells also produce and are exposed to these physical forces. Composed of a combination of tension and compression, these forces are significant in tumors, but negligible in most normal tissues. Methods and Results: We developed the experimental and mathematical frameworks to provide (i) two-dimensional spatial map of solid stress in tumors (planar cut method), (ii) sensitive estimation of solid stress in small tumors with small magnitudes of solid stress, e.g., metastatic lesions (slicing method), and (iii) in situ quantification of solid stress in tumors, which retains the effects of the normal surrounding tissues (needle biopsy method). All three methods are based on the physical concept of releasing the solid stress in a controlled way with defined geometry, and then quantifying the stress-induced deformation by high-resolution ultrasonography or optical microscopy. Given the specific topography of the stress relaxation and the geometric and material properties of the tumour, the solid stress and discharged elastic energy is estimated using mathematical modeling. Applying these novel methods to multiple mouse cancer models in primary and metastatic settings has led to the following novel findings: (i) solid stress and elastic energy may be different between primary vs. metastatic settings, as they depend on both cancer cells and their microenvironment; (ii) tumor with higher elastic energy are not necessarily stiffer, and the stiffer tumors do not necessarily have higher elastic energy; (iii) solid stress increases with tumour size; and (iv) the normal tissue surrounding a tumour significantly contributes to the intratumoral solid stress. Conclusions: We developed three distinct methods to perform in situ and sensitive measurement of solid stress and obtain 2-D spatial map of solid stress in human and mouse tumors. Application of these methods in models of primary tumors and metastasis revealed that: (i) solid stress depends on both cancer cells and their microenvironment; it increases with tumour size; and mechanical confinement by the surrounding tissue substantially contributes to intratumoral solid stress. Further study of the genesis and consequences of solid stress, facilitated by the engineering principles presented here, may lead to significant discoveries and new therapies. Citation Format: Hadi Nia, Hao Liu, Giorgio Seano, Meenal Datta, Dennis Jones, Nuh Rahbari, Joao Incio, Vikash Chauhan, Keehoon Jung, John Martin, Vasileios Askoxylakis, Tim Padera, Dai Fukumura, Yves Boucher, Francis Hornicek, Alan Grodzinsky, James Baish, Lance Munn, Rakesh Jain. Solid stress and elastic energy as measures of tumor mechanopathology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3987. doi:10.1158/1538-7445.AM2017-3987

Abstract It remains unclear how obesity worsens treatment outcomes in patients with pancreatic ductal adenocarcinoma (PDAC). In normal pancreas, obesity promotes inflammation and fibrosis. We found in mouse models of PDAC that obesity also promotes desmoplasia associated with accelerated tumor growth and impaired delivery/efficacy of chemotherapeutics through reduced perfusion. Genetic and pharmacologic inhibition of angiotensin-II type-1 receptor reverses obesity-augmented desmoplasia and tumor growth and improves response to chemotherapy. Augmented activation of pancreatic stellate cells (PSC) in obesity is induced by tumor-associated neutrophils (TAN) recruited by adipocyte-secreted IL1β. PSCs further secrete IL1β, and inactivation of PSCs reduces IL1β expression and TAN recruitment. Furthermore, depletion of TANs, IL1β inhibition, or inactivation of PSCs prevents obesity-accelerated tumor growth. In patients with pancreatic cancer, we confirmed that obesity is associated with increased desmoplasia and reduced response to chemotherapy. We conclude that cross-talk between adipocytes, TANs, and PSCs exacerbates desmoplasia and promotes tumor progression in obesity. Significance: Considering the current obesity pandemic, unraveling the mechanisms underlying obesity-induced cancer progression is an urgent need. We found that the aggravation of desmoplasia is a key mechanism of obesity-promoted PDAC progression. Importantly, we discovered that clinically available antifibrotic/inflammatory agents can improve the treatment response of PDAC in obese hosts. Cancer Discov; 6(8); 852–69. ©2016 AACR. See related commentary by Bronte and Tortora, p. 821. This article is highlighted in the In This Issue feature, p. 803

Solid stress and tissue stiffness affect tumour growth, invasion, metastasis and treatment. Unlike stiffness, which can be precisely mapped in tumours, the measurement of solid stresses is challenging. Here, we show that 2D spatial maps of the solid stress and the resulting elastic energy in excised or in situ tumours with arbitrary shapes and a wide range of sizes can be obtained via three distinct and quantitative techniques that rely on the measurement of tissue displacement after disruption of the confining structures. Application of these methods in models of primary tumours and metastasis revealed that (i) solid stress depends on both cancer cells and their microenvironments, (ii) solid stress increases with tumour size and (iii) mechanical confinement by the surrounding tissue substantially contributes to intratumoral solid stress. Further study of the genesis and consequences of solid stress, facilitated by the engineering principles presented here, may lead to new discoveries and therapies.

Impaired perfusion is a hallmark of solid tumors that promotes progression, immunosuppression, and treatment resistance and is partly caused by vascular compression from excessive extravascular stresses (Chauhan et al., 2013Chauhan V.P. Martin J.D. Liu H. Lacorre D.A. Jain S.R. Kozin S.V. Stylianopoulos T. Mousa A.S. Han X. Adstamongkonkul P. et al.Nat. Commun. 2013; 4: 2516Crossref PubMed Scopus (606) Google Scholar, Stylianopoulos et al., 2012Stylianopoulos T. Martin J.D. Chauhan V.P. Jain S.R. Diop-Frimpong B. Bardeesy N. Smith B.L. Ferrone C.R. Hornicek F.J. Boucher Y. et al.Proc. Natl. Acad. Sci. USA. 2012; 109: 15101-15108Crossref PubMed Scopus (542) Google Scholar). The extravascular stress exerted by fluid is referred to as interstitial fluid pressure (IFP) and that by solid components as solid stress (SS). IFP is near zero in most tissues, but rises in tumors as impaired lymphatics fail to drain fluid leaking from blood vessels and IFP equilibrates with microvascular pressure (MVP) (Boucher and Jain, 1992Boucher Y. Jain R.K. Cancer Res. 1992; 52: 5110-5114PubMed Google Scholar). Due to equilibration, IFP in tumors can only transiently exceed MVP and thus cannot compress tumor vessels (Figure S1A available online). SS is generated as cells push and pull on their surroundings during proliferation and migration and is transmitted by extracellular matrix (Stylianopoulos et al., 2012Stylianopoulos T. Martin J.D. Chauhan V.P. Jain S.R. Diop-Frimpong B. Bardeesy N. Smith B.L. Ferrone C.R. Hornicek F.J. Boucher Y. et al.Proc. Natl. Acad. Sci. USA. 2012; 109: 15101-15108Crossref PubMed Scopus (542) Google Scholar). SS is greatly and chronically elevated in tumors due to high cell and matrix densities and can compress blood vessels (Figure S1A). Since the causes and consequences of elevated IFP and SS are different, strategies for alleviating these mechanical stresses are likely to be distinct. In their article, Provenzano et al. elegantly showed that hyaluronan (HA) can mechanically compress blood vessels in pancreatic ductal adenocarcinoma (PDA) (Provenzano et al., 2012Provenzano P.P. Cuevas C. Chang A.E. Goel V.K. Von Hoff D.D. Hingorani S.R. Cancer Cell. 2012; 21: 418-429Abstract Full Text Full Text PDF PubMed Scopus (1407) Google Scholar). However, their proposed mechanism—that HA leads to very high IFP that collapses vessels—is not consistent with the physiology of fluid homeostasis and calls for careful assessment of IFP in PDAs. They suggest that mean IFP can reach 99 mmHg (range 75–130 mmHg), presumably higher than MVP, in the Pdx1-Cre/KrasLSL-G12D/+/p53LSL-R172H/+ (KPC) PDA model based on measurements made with a piezoelectric probe. To evaluate this, we measured IFP in KPC tumors with the wick-in-needle technique—which has been validated against the gold-standard micropipette technique (Boucher and Jain, 1992Boucher Y. Jain R.K. Cancer Res. 1992; 52: 5110-5114PubMed Google Scholar). We further measured IFP in additional PDA models, Ptf1-Cre/KrasLSL-G12D/+/p53L/+ (KPdC) and Ptf1-Cre/ROSA26-LSL-rtTA-IRES-GFP/KrasTetO-LSL-G12D/+/p53L/+ (iKPdC), which highly express HA (Figure S1B). The mean IFP was 8.1 mmHg (range 4.7–10.9 mmHg) in KPC, 3.4 mmHg (range 1.6–5.6 mmHg) in KPdC, and 6.7 mmHg (range 6.1–8.0 mmHg) in iKPdC (Figure S1C)—over an order of magnitude lower than the IFP levels reported by Provenzano et al. We also measured IFP with wick-in-needle in the tumors of four treatment-naive PDA patients (Figure S1C), and the mean IFP was 11.8 mmHg (range 6.1–16.6 mmHg). As these IFPs do not exceed typical MVPs, IFP cannot compress PDA vessels, leaving SS as the primary cause. Provenzano et al. also propose that PDA IFP is not driven by equilibration with MVP because PDA vessels are nonleaky, i.e., nonpermeable to macromolecules. As evidence, they state that PDA IFP measured with the piezeoelectric probe remains elevated upon cardiac cessation, indicating that blood pressure is not driving IFP. With wick-in-needle, we found that cardiac cessation reduced IFP to zero in all three PDA models, confirming that blood pressure drives elevated IFP (Boucher and Jain, 1992Boucher Y. Jain R.K. Cancer Res. 1992; 52: 5110-5114PubMed Google Scholar). Furthermore, their hypothesis that PDA vessels are nonpermeable to macromolecules is contradicted by their own data—PEGylated recombinant human hyaluronidase (PEGPH20), a macromolecule, clearly permeates across PDA vessels since it acts on interstitial HA. Moreover, the efficacy in PDA patients of nanoparticle-albumin-bound-paclitaxel, an FDA-approved macromolecule, also indicates that PDA vessels are somewhat leaky. We conclude that PDA IFP is indeed driven by blood pressure and that fluid exchange between the intravascular and interstitial space in PDA facilitates equilibration of IFP and MVP as in other tumors. The discrepancy between our IFP measurements and those of Provenzano et al. may stem from their use of the piezoelectric probe technique (Ozerdem and Hargens, 2005Ozerdem U. Hargens A.R. Microvasc. Res. 2005; 70: 116-120Crossref PubMed Scopus (63) Google Scholar), which we believe suffers from artifacts coming from SS. As evidence for our hypothesis, Provenzano et al. measured an IFP of 10.4 mmHg (range 8–13 mmHg) in normal mouse pancreata, although normal murine tissues typically have slightly negative IFPs. Ozerdem and Hargens tested this technique against wick-in-needle in a single tumor model in two mice, but they did not carefully test for such artifacts—for example by comparison to wick-in-needle in tissues of varying matrix or cell density. We therefore compared the piezoelectric probe technique to wick-in-needle in multiple normal murine tissues. We found that the piezoelectric probe produces significantly higher measurements when compared with wick-in-needle (Figures S1D and S1E). For example, we measured pancreas IFP as −0.7 mmHg (range −0.9 to −0.5 mmHg) with wick-in-needle, whereas our pancreas measurements with the piezoelectric probe were 9.8 mmHg (range 8.5–11.3 mmHg). This discrepancy most likely occurs because the sensor in the piezoelectric probe, unlike that in wick-in-needle, directly contacts cells and matrix allowing solid tissue components to contribute to the reading. We conclude that the piezoelectric probe method developed by Ozerdem and Hargens—and used by Provenzano et al.—measures pressures that are higher than the actual IFP due to artifacts from solid tissue components. As demonstrated here, HA-rich desmoplasia in PDA does not produce unusually high IFP, IFP cannot compress PDA vessels, and the technique Provenzano et al. used to measure IFP actually measures a combination of IFP and SS. Meanwhile, we found that HA increases SS through storage and transmission mechanisms (Stylianopoulos et al., 2012Stylianopoulos T. Martin J.D. Chauhan V.P. Jain S.R. Diop-Frimpong B. Bardeesy N. Smith B.L. Ferrone C.R. Hornicek F.J. Boucher Y. et al.Proc. Natl. Acad. Sci. USA. 2012; 109: 15101-15108Crossref PubMed Scopus (542) Google Scholar). Thus, PEGPH20 most likely reduces SS, thereby decompressing vessels. Since IFP cannot compress blood vessels, we propose that the mechanism of vessel decompression by PEGPH20 is solely a reduction in SS. Importantly, therapies that alleviate SS cause decompression of vessels and improve PDA treatment (Chauhan et al., 2013Chauhan V.P. Martin J.D. Liu H. Lacorre D.A. Jain S.R. Kozin S.V. Stylianopoulos T. Mousa A.S. Han X. Adstamongkonkul P. et al.Nat. Commun. 2013; 4: 2516Crossref PubMed Scopus (606) Google Scholar). Thus, we concur that PEGPH20 has immense promise for PDA, and we hope that this correspondence clarifies its mechanism. Download .pdf (.27 MB) Help with pdf files Document S1. Supplemental Experimental Procedures and Figure S1 Response to Chauhan et al.: Interstitial Pressure and Vascular Collapse in Pancreas Cancer—Fluids and Solids, Measurement and MeaningDelGiorno et al.Cancer CellJuly 14, 2014In BriefChauhan et al. suggest that vascular collapse and hypoperfusion in pancreatic ductal adenocarcinoma (PDA) are caused by solid stress (SS) (Chauhan et al., 2014) instead of the elevated interstitial fluid pressure (IFP) associated with high extravascular concentrations of hyaluronan (Provenzano et al., 2012). We appreciate their attention to our work and the opportunity to clarify underlying mechanisms. Chauhan et al. make four important claims, to which we respond. Full-Text PDF Open Archive

Cancer nanomedicines approved so far minimize toxicity, but their efficacy is often limited by physiological barriers posed by the tumour microenvironment. Here, we discuss how these barriers can be overcome through innovative nanomedicine design and through creative manipulation of the tumour microenvironment.