Vikash P. Chauhan

Multistage nanoparticle delivery system for deep penetration into tumor tissue

Current Food and Drug Administration-approved cancer nanotherapeutics, which passively accumulate around leaky regions of the tumor vasculature because of an enhanced permeation and retention (EPR) effect, have provided only modest survival benefits. This suboptimal outcome is likely due to physiological barriers that hinder delivery of the nanotherapeutics throughout the tumor. Many of these nanotherapeutics are ≈100 nm in diameter and exhibit enhanced accumulation around the leaky regions of the tumor vasculature, but their large size hinders penetration into the dense collagen matrix. Therefore, we propose a multistage system in which 100-nm nanoparticles “shrink” to 10-nm nanoparticles after they extravasate from leaky regions of the tumor vasculature and are exposed to the tumor microenvironment. The shrunken nanoparticles can more readily diffuse throughout the tumors interstitial space. This size change is triggered by proteases that are highly expressed in the tumor microenvironment such as MMP-2, which degrade the cores of 100-nm gelatin nanoparticles, releasing smaller 10-nm nanoparticles from their surface. We used quantum dots (QD) as a model system for the 10-nm particles because their fluorescence can be used to demonstrate the validity of our approach. In vitro MMP-2 activation of the multistage nanoparticles revealed that the size change was efficient and effective in the enhancement of diffusive transport. In vivo circulation half-life and intratumoral diffusion measurements indicate that our multistage nanoparticles exhibited both the long circulation half-life necessary for the EPR effect and the deep tumor penetration required for delivery into the tumors dense collagen matrix.

Compact high-quality CdSe–CdS core–shell nanocrystals with narrow emission linewidths and suppressed blinking

High particle uniformity, high photoluminescence quantum yields, narrow and symmetric emission spectral lineshapes and minimal single-dot emission intermittency (known as blinking) have been recognized as universal requirements for the successful use of colloidal quantum dots in nearly all optical applications. However, synthesizing samples that simultaneously meet all these four criteria has proven challenging. Here, we report the synthesis of such high-quality CdSe-CdS core-shell quantum dots in an optimized process that maintains a slow growth rate of the shell through the use of octanethiol and cadmium oleate as precursors. In contrast with previous observations, single-dot blinking is significantly suppressed with only a relatively thin shell. Furthermore, we demonstrate the elimination of the ensemble luminescence photodarkening that is an intrinsic consequence of quantum dot blinking statistical ageing. Furthermore, the small size and high photoluminescence quantum yields of these novel quantum dots render them superior in vivo imaging agents compared with conventional quantum dots. We anticipate these quantum dots will also result in significant improvement in the performance of quantum dots in other applications such as solid-state lighting and illumination.

Strategies for advancing cancer nanomedicine

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.

Delivery of molecular and nanoscale medicine to tumors: transport barriers and strategies.

Tumors are similar to organs, with unique physiology giving rise to an unusual set of transport barriers to drug delivery. Cancer therapy is limited by nonuniform drug delivery via blood vessels, inhomogeneous drug transport into tumor interstitium from the vascular compartment, and hindered transport through tumor interstitium to the target cells. Four major abnormal physical and physiological properties contribute to these transport barriers. Accumulated solid stress compresses blood vessels to diminish the drug supply to many tumor regions. Immature vasculature with high viscous and geometric resistances and reduced pressure gradients leads to sluggish and heterogeneous blood flow in tumors to further limit drug supply. Nonfunctional lymphatics coupled with highly permeable blood vessels result in elevated hydrostatic pressure in tumors to abrogate convective drug transport from blood vessels into and throughout most of the tumor tissue. Finally, a dense structure of interstitial matrix and cells serves as a tortuous, viscous, and steric barrier to diffusion of therapeutic agents. In this review, we discuss the origins and implications of these barriers. We then highlight strategies for overcoming these barriers by modulating either drug properties or the tumor microenvironment itself to enhance the delivery and effectiveness of drugs in tumors.

Angiotensin inhibition enhances drug delivery and potentiates chemotherapy by decompressing tumour blood vessels

Cancer and stromal cells actively exert physical forces (solid stress) to compress tumour blood vessels, thus reducing vascular perfusion. Tumour interstitial matrix also contributes to solid stress, with hyaluronan implicated as the primary matrix molecule responsible for vessel compression because of its swelling behaviour. Here we show, unexpectedly, that hyaluronan compresses vessels only in collagen-rich tumours, suggesting that collagen and hyaluronan together are critical targets for decompressing tumour vessels. We demonstrate that the angiotensin inhibitor losartan reduces stromal collagen and hyaluronan production, associated with decreased expression of profibrotic signals TGF-β1, CCN2 and ET-1, downstream of angiotensin-II-receptor-1 inhibition. Consequently, losartan reduces solid stress in tumours resulting in increased vascular perfusion. Through this physical mechanism, losartan improves drug and oxygen delivery to tumours, thereby potentiating chemotherapy and reducing hypoxia in breast and pancreatic cancer models. Thus, angiotensin inhibitors —inexpensive drugs with decades of safe use — could be rapidly repurposed as cancer therapeutics.

Causes, consequences, and remedies for growth-induced solid stress in murine and human tumors

The presence of growth-induced solid stresses in tumors has been suspected for some time, but these stresses were largely estimated using mathematical models. Solid stresses can deform the surrounding tissues and compress intratumoral lymphatic and blood vessels. Compression of lymphatic vessels elevates interstitial fluid pressure, whereas compression of blood vessels reduces blood flow. Reduced blood flow, in turn, leads to hypoxia, which promotes tumor progression, immunosuppression, inflammation, invasion, and metastasis and lowers the efficacy of chemo-, radio-, and immunotherapies. Thus, strategies designed to alleviate solid stress have the potential to improve cancer treatment. However, a lack of methods for measuring solid stress has hindered the development of solid stress-alleviating drugs. Here, we present a simple technique to estimate the growth-induced solid stress accumulated within animal and human tumors, and we show that this stress can be reduced by depleting cancer cells, fibroblasts, collagen, and/or hyaluronan, resulting in improved tumor perfusion. Furthermore, we show that therapeutic depletion of carcinoma-associated fibroblasts with an inhibitor of the sonic hedgehog pathway reduces solid stress, decompresses blood and lymphatic vessels, and increases perfusion. In addition to providing insights into the mechanopathology of tumors, our approach can serve as a rapid screen for stress-reducing and perfusion-enhancing drugs.

Losartan inhibits collagen I synthesis and improves the distribution and efficacy of nanotherapeutics in tumors

The dense collagen network in tumors significantly reduces the penetration and efficacy of nanotherapeutics. We tested whether losartan—a clinically approved angiotensin II receptor antagonist with noted antifibrotic activity—can enhance the penetration and efficacy of nanomedicine. We found that losartan inhibited collagen I production by carcinoma-associated fibroblasts isolated from breast cancer biopsies. Additionally, it led to a dose-dependent reduction in stromal collagen in desmoplastic models of human breast, pancreatic, and skin tumors in mice. Furthermore, losartan improved the distribution and therapeutic efficacy of intratumorally injected oncolytic herpes simplex viruses. Finally, it also enhanced the efficacy of i.v. injected pegylated liposomal doxorubicin (Doxil). Thus, losartan has the potential to enhance the efficacy of nanotherapeutics in patients with desmoplastic tumors.

Obesity-induced inflammation and desmoplasia promote pancreatic cancer progression and resistance to chemotherapy

UNLABELLED 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. 821This article is highlighted in the In This Issue feature, p. 803.

Compression of pancreatic tumor blood vessels by hyaluronan is caused by solid stress and not interstitial fluid pressure.

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., 2013; Stylianopoulos et al., 2012). 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, 1992). Due to equilibration, IFP in tumors can only transiently exceed MVP and thus cannot compress tumor vessels (Figure S1A). SS is generated as cells push and pull on their surroundings during proliferation and migration, and is transmitted by extracellular matrix (Stylianopoulos et al., 2012). 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., 2012). 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 using a piezoelectric-probe. To evaluate this, we measured IFP in KPC tumors (Figure S1B) with the wick-in-needle technique—which has been validated against the gold-standard micropipette technique (Boucher and Jain, 1992). 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 S1E). 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—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 S1B), and the mean IFP was 11.8 mmHg (range 6.1–16.6 mmHg). As these IFPs are in the range of typical MVPs, our established concept holds: SS compresses PDA vessels while IFP cannot. Provenzano et al. also propose that PDA IFP is not driven by equilibration with MVP because PDA vessels are non-leaky, i.e. non-permeable 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, 1992). Furthermore, their hypothesis that PDA vessels are non-permeable to macromolecules is contradicted by their own data—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, 2005), which we believe suffers from artifacts coming from SS. As strong 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 comparing 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 (Figure S1C, D). For example, we measured pancreas IFP as -0.7 mmHg (range -0.9 – -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 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., 2012). Thus PEGPH20 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 decompress vessels and improve PDA treatment (Chauhan et al., 2013). Thus we concur that PEGPH20 has immense promise for PDA, and we hope that this Correspondence clarifies its mechanism.

Multiscale Measurements Distinguish Cellular and Interstitial Hindrances to Diffusion In Vivo

Molecular cancer therapy relies on interstitial diffusion for drug distribution in solid tumors. A mechanistic understanding of how tumor components affect diffusion is necessary to advance cancer drug development. Yet, because of limitations in current techniques, it is unclear how individual tissue components hinder diffusion. We developed multiscale fluorescence recovery after photobleaching (MS-FRAP) to address this deficiency. Diffusion measurements facilitated by MS-FRAP distinguish the diffusive hindrance of the interstitial versus cellular constituents in living tissue. Using multiscale diffusion measurements in vivo, we resolved the contributions of these two major tissue components toward impeding diffusive transport in solid tumors and subcutaneous tissue in mice. We further used MS-FRAP in interstitial matrix-mimetic gels and in vivo to show the influence of physical interactions between collagen and hyaluronan on diffusive hindrance through the interstitium. Through these studies, we show that interstitial hyaluronan paradoxically improves diffusion and that reducing cellularity enhances diffusive macromolecular transport in solid tumors.