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.publishedVersio

High Interstitial Fluid Pressure Is Associated with Tumor-Line Specific Vascular Abnormalities in Human Melanoma Xenografts

PURPOSE: Interstitial fluid pressure (IFP) is highly elevated in many solid tumors. High IFP has been associated with low radiocurability and high metastatic frequency in human melanoma xenografts and with poor survival after radiation therapy in cervical cancer patients. Abnormalities in tumor vascular networks have been identified as an important cause of elevated tumor IFP. The aim of this study was to investigate the relationship between tumor IFP and the functional and morphological properties of tumor vascular networks. MATERIALS AND METHODS: A-07-GFP and R-18-GFP human melanomas growing in dorsal window chambers in BALB/c nu/nu mice were used as preclinical tumor models. Functional and morphological parameters of the vascular network were assessed from first-pass imaging movies and vascular maps recorded after intravenous bolus injection of 155-kDa tetramethylrhodamine isothiocyanate-labeled dextran. IFP was measured in the center of the tumors using a Millar catheter. Angiogenic profiles of A-07-GFP and R-18-GFP cells were obtained with a quantitative PCR array. RESULTS: High IFP was associated with low growth rate and low vascular density in A-07-GFP tumors, and with high growth rate and high vascular density in R-18-GFP tumors. A-07-GFP tumors showed chaotic and highly disorganized vascular networks, while R-18-GFP tumors showed more organized vascular networks with supplying arterioles in the tumor center and draining venules in the tumor periphery. Furthermore, A-07-GFP and R-18-GFP cells differed substantially in angiogenic profiles. A-07-GFP tumors with high IFP showed high geometric resistance to blood flow due to high vessel tortuosity. R-18-GFP tumors with high IFP showed high geometric resistance to blood flow due to a large number of narrow tumor capillaries. CONCLUSIONS: High IFP in A-07-GFP and R-18-GFP human melanoma xenografts was primarily a consequence of high blood flow resistance caused by tumor-line specific vascular abnormalities

HPV-test i screeningprogrammet mot livmorhalskreft for aldersgruppen 25-33 år: systematisk oversikt

Introduction Prolonged infections with human papillomavirus (HPV) have a causative role in 99% of cervical cancer cases. The majority of HPV infections is nevertheless defeated by the immune system without causing cervical cancer. Since 1995, Norwegian women in the age group of 25 to 69 years have been invited to a national screening program for cervical cancer. The screening program has used conventional cytology to assess cell samples, but from 2015 primary HPV-testing has gradually been introduced for the age group of 34 to 69 years. This implementation was completed first quarter 2022. Also in 2022, the first cohort that was offered the HPV vaccine in the national vaccine program will be invited to participate in the national screening program for cervical cancer. Objective The objective of this systematic review was to investigate the effect of combinations of HPV-test and cytology in screening programs for cervical cancer for women aged 25-33 years. We intended to evaluate three interventions: (1) replace primary cytological evaluation with primary HPV-test, (2) use both HPV-test and cytology (cotesting), and (3) use HPV-test as quality assurance for cytology. Separate analyses and assessments were required for HPV-vaccinated and unvaccinated women. Method We conducted a systematic literature search in relevant databases. Two researchers assessed titles, abstracts, and full text of relevant references against the selection criteria. We used EPPI-Reviewer in the selection process, including various machine learning functionalities to streamline the selection process. One researcher extracted and analysed data from the included studies and another checked the data extraction. Two researchers assessed the methodological quality with validated checklists and tools and assessed our confidence in the results using GRADE. We also assessed the transferability to the Norwegian context. Results We included six randomized trials. Four trials investigated the effect of intervention 1 and two investigated the effect of intervention 2. We found no studies evaluating intervention 3. All trials were conducted in national or regional screening programs in high-income countries (in Australia, Sweden, Finland, Netherlands, Italy, and Canada). One trial investigated HPV-vaccinated women, and five trials investigated unvaccinated women. The trials investigated broad age groups, but also reported separate data for the age group that was relevant for this review (25-33 years) and/or for relevant age subgroups (25-29 years or 30-33 years). For HPV-vaccinated women, we found that primary HPV-test possibly results in more referrals to colposcopy and more findings of CIN2+ and CIN3+ than primary cytology in the first round of screening (Summary of findings 1). For unvaccinated women, we found that primary HPV-test compared to primary cytology probably results in more referrals to colposcopy and more findings of CIN2+ and CIN3+ in the first round of screening (Summary of findings 1), and probably less findings of CIN2+ and possibly less findings of CIN3+ in the second round of screening. The effects in the first round of screening were observed in both subgroups (25-29 years and 30-33 years) but were more pronounced for the youngest subgroup. Summary of findings 1. Primary HPV-test compared to primary cytology for HPV-vaccinated and unvaccinated women aged 25-33 years Outcome Anticipated absolute effects (95% CI) Relative effect (95% CI) Number of participants Certainty (GRADE) Primary cytology Primary HPV-test HPV-vaccinated women CIN2+ 5 per 1 000 26 per 1 000 (3 to 202) RR 5.55 (0.72 to 42.7) 629 ⨁⨁◯◯ Low a CIN3+ 5 per 1 000 22 per 1 000 (3 to 169) RR 4.54 (0.58 to 35.6) 629 ⨁⨁◯◯ Low a Colposcopy 47 per 1 000 82 per 1 000 (41 to 162) RR 1.72 (0.86 to 3.41) 629 ⨁⨁◯◯ Low a Unvaccinated women CIN2+ 21 per 1 000 25 per 1 000 (22 to 29) RR 1.18 (1.03 to 1.36) 35 234 ⨁⨁⨁◯ Moderate b CIN3+ 14 per 1 000 22 per 1 000 (14 to 34) RR 1.50 (0.96 to 2.35) 4 849 ⨁⨁⨁◯ Moderate c Colposcopy 40 per 1 000 58 per 1 000 (44 to 77) RR 1.45 (1.10 to 1.92) 35 234 ⨁⨁⨁◯ Moderate b a) Extremely broad confidence interval including no effect and both positive and negative effects. Less participants than the optimal information size. We have downregulated by two units. b) One of the studies has high risk of bias. We have downregulated by one unit. c) Broad confidence interval including large and small effects, and few participants. We have downregulated by one unit. The trials investigating intervention 2 evaluated unvaccinated women. We found that HPV-test and cytologi (cotesting) compared to cytology alone possibly results in more referrals to colposcopy and more findings of CIN2+ in the first round of screening (Summary of findings 2), and possibly less findings of CIN2+ in the second round of screening. The evidence for CIN3+ was highly uncertain for this comparison (Summary of findings 2). None of the included studies reported referrals to conization, adverse effects after conization, overtreatment, or quality of life. Summary of findings 2. HPV-test and cytology (cotesting) compared to cytology for unvaccinated women aged 25-33 years Outcome Anticipated absolute effects (95% CI) Relative effect (95% CI) Number of participants Certainty (GRADE) Cytology Cotesting CIN2+ 14 per 1 000 20 per 1 000 (12 to 32) RR 1.43 (0.89 to 2.29) 18 677 ⨁⨁◯◯ Low a CIN3+ 9 per 1 000 10 per 1 000 (3 to 169) RR 1.07 (0.80 to 1.43) 18 677 ⨁◯◯◯ Very low a,b Colposcopy 41 per 1 000 48 per 1 000 (41 to 162) RR 1.17 (0.93 to 1.48) 6 267 ⨁⨁◯◯ Low a a) Broad confidence interval including no effect and both positive and negative effects. We have downregulated by two units. b) The included studies show opposite effects. We have downregulated by one unit. Discussion The evidence in this review addresses intervention 1 and intervention 2, but studies evaluating intervention 3 were not found. All the included trials were conducted in national or regional screening programs in high-income countries and should be transferable to the Norwegian context. Most trials evaluating intervention 1 used the test that Norwegian women below 34 years are offered in the national screening program as comparator. This comparison is thus directly transferable to the Norwegian practice. The trials evaluating intervention 2 used cytology alone as comparator, and it is unclear to what extent this intervention would compare to the test women below 34 years are offered in the Norwegian national screening program. Only one trial investigated HPV-vaccinated women, and the number of participants in this trial was low. The evidence for HPV-vaccinated women is thus substantially weaker than for unvaccinated women. Novel studies investigating the effect of screening strategies for HPV-vaccinated women are thus needed. This need is urgent because the first cohort of women that were offered the HPV-vaccine in the Norwegian vaccine program are invited to the national screening program for cervical cancer in 2022. Conclusion Replacing primary cytology with primary HPV-test probably results in more referrals to colposcopy and earlier detection of CIN2+ and CIN3+ for unvaccinated women below 34 years. HPV-test and cytology (cotesting) possibly results in more referrals to colposcopy and earlier detection of CIN2+ than cytology alone. The evidence for HPV-vaccinated women is substantially weaker than for unvaccinated women. Primary HPV-test possibly results in more referrals to colposcopy and earlier detection of CIN2+ and CIN3+ than primary cytology, but novel studies investigating effects of screening strategies for HPV-vaccinated women are needed

Temporal Heterogeneity in Blood Supply in Human Tumor Xenografts12

Temporal heterogeneities in tumor blood supply were studied by using a recently developed first-pass imaging technique. First-pass imaging movies of A-07-GFP human tumor xenografts growing in window chambers were recorded at a frame rate of ∼9 fps and a spatial resolution of 10.8 x 10.8 µm2 after a bolus of 155-kDa tetramethylrhodamine isothiocyanate-labeled dextran had been administered intravenously. Each tumor was subjected to imaging thrice, with 20 minutes between each repetition. Highly specific maps of the vascular network and blood supply time (BST) images (i.e., images of the time from when arterial blood enters a tumor through the main supplying artery until it reaches a vessel segment within the tumor) were produced from the movies. The tumors had one to three supplying arterioles and showed substantial temporal heterogeneity in BST. Homogeneous changes in BST in the entire vascular network were seen in tumors supplied by one arteriole. Blood supply time fluctuations in tumor subregions were observed in tumors having two or three supplying arterioles. In addition, individual vessel segments frequently showed significant changes in BST with time. High-magnification transmission microscopy imaging substantiated that BST changes could be a consequence of arterial/arteriolar vasomotor activity, vessel wall compression, varying flow rate, and vascular stasis

Sunitinib treatment does not improve blood supply but induces hypoxia in human melanoma xenografts

Abstract Background Antiangiogenic agents that disrupt the vascular endothelial growth factor pathway have been demonstrated to normalize tumor vasculature and improve tumor oxygenation in some studies and to induce hypoxia in others. The aim of this preclinical study was to investigate the effect of sunitinib treatment on the morphology and function of tumor vasculature and on tumor oxygenation. Methods A-07-GFP and R-18-GFP human melanoma xenografts grown in dorsal window chambers were used as preclinical tumor models. Morphologic parameters of tumor vascular networks were assessed from high-resolution transillumination images, and tumor blood supply time was assessed from first-pass imaging movies recorded after a bolus of 155 kDa tetramethylrhodamine isothiocyanate-labeled dextran had been administered intravenously. Tumor hypoxia was assessed from immunohistochemical preparations of the imaged tissue by use of pimonidazole as a hypoxia marker. Results Sunitinib treatment reduced vessel densities, increased vessel segment lengths, did not affect blood supply times, and increased hypoxic area fractions. Conclusion Sunitinib treatment did not improve vascular function but induced hypoxia in A-07-GFP and R-18-GFP tumors.</p

The Effect of Sunitinib Treatment in Human Melanoma Xenografts: Associations with Angiogenic Profiles

The effect of antiangiogenic agents targeting the vascular endothelial growth factor A (VEGF-A) pathway has been reported to vary substantially in preclinical studies. The purpose of this study was to investigate the effect of sunitinib treatment on tumor vasculature and oxygenation in melanoma xenografts with different angiogenic profiles. A-07, U-25, D-12, or R-18 melanoma xenografts were grown in dorsal window chambers and given daily treatments of sunitinib (40 mg/kg) or vehicle. Morphologic parameters of tumor vascular networks were assessed from high-resolution transillumination images, and tumor blood supply times (BSTs) were assessed from first-pass imaging movies. Tumor hypoxia was assessed with immunohistochemistry by using pimonidazole as hypoxia marker, and the gene expression and the protein secretion rate of angiogenic factors were assessed by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The melanoma lines differed substantially in the expression of VEGF-A, VEGF-C, and platelet-derived growth factor A. Sunitinib treatment reduced vessel densities and induced hypoxia in all melanoma lines, and the magnitude of the effect was associated with the gene expression and protein secretion rate of VEGF-A. Sunitinib treatment also increased vessel segment lengths, reduced the number of small-diameter vessels, and inhibited growth-induced increases in the diameter of surviving vessels but did not change BST. In conclusion, sunitinib treatment did not improve vascular function but reduced vessel density and induced hypoxia in human melanoma xenografts. The magnitude of the treatment-induced effect was associated with the VEGF-A expression of the melanoma lines

Vascular abnormalities and development of hypoxia in microscopic melanoma xenografts

Abstract Background Studies investigating the oxygenation status and the development of hypoxia in microscopic tumors are sparse. The purpose of this study was to measure the extent of hypoxia in microscopic melanoma xenografts and to search for possible mechanisms leading to the development of hypoxia in these tumors. Methods A-07, D-12, R-18, and U-25 human melanoma xenografts grown in dorsal window chambers or as flank tumors were used as preclinical tumor models. Morphologic and functional parameters of vascular networks were assessed with intravital microscopy, and the expression of angiogenesis-related genes was assessed with quantitative PCR. Microvessels, pericytes, and the extent of hypoxia were assessed by immunohistochemistry in microscopic tumors by using CD31, αSMA, and pimonidazole as markers, and the extent of radiobiological hypoxia was assessed in macroscopic flank tumors. Results Macroscopic R-18 and U-25 tumors showed extensive hypoxia, whereas macroscopic A-07 and D-12 tumors were less hypoxic. R-18 and U-25 tumors developed hypoxic regions before they reached a size of 2–3 mm in diameter, whereas A-07 and D-12 tumors of similar size did not show hypoxic regions. The development of hypoxic regions was not caused by low vessel density, but was rather a result of inadequate vascular function. Inadequate vascular function was not caused by low vessel diameters or long vessel segments, but was associated with poor vascular pericyte coverage. Poor pericyte coverage was associated with the expression of eight angiogenesis-related genes. Conclusions Two of the four investigated melanoma models developed hypoxic regions in microscopic tumors, and the development of hypoxia was associated with poor vascular pericyte coverage and inadequate vascular function

Antiangiogenic agents targeting different angiogenic pathways have opposite effects on tumor hypoxia in R-18 human melanoma xenografts

Abstract Background Studies comparing the effect of antiangiogenic agents targeting different angiogenic pathways are sparse. The purpose of this study was to compare the effect of properdistatin and sunitinib treatment in a preclinical model of malignant melanoma. Properdistatin is a small peptide derived from the thrombospondin-1 domain of the plasma protein properdin, and sunitinib is a tyrosine kinase inhibitor targeting several receptors including the vascular endothelial growth factor receptors. Methods R-18 human melanoma xenografts growing in dorsal window chambers were treated with properdistatin, sunitinib, or vehicle. Parameters describing the morphology of tumor vasculature were assessed from high-resolution transillumination images, and BST (blood supply time; the time needed for arterial blood to flow from the main supplying artery to downstream microvessels) was assessed from first-pass imaging movies recorded after a bolus of fluorescence-labeled dextran had been administered intravenously. Tumor hypoxia was assessed from immunohistochemical preparations of the imaged tissue by using pimonidazole as a hypoxia marker. Results Properdistatin treatment selectively removed small-diameter vessels and reduced BST, whereas sunitinib treatment reduced the density of small- and large-diameter vessel similarly and did not change BST. These observations imply that properdistatin treatment reduced geometric resistance to blood flow and improved vascular function, whereas sunitinib treatment did not affect vascular function. Accordingly, sunitinib-treated tumors showed higher hypoxic fractions than properdistatin-treated tumors. Conclusions Properdistatin and sunitinib both inhibited angiogenesis, but had distinctly different effects on vascular morphology, vascular function, and extent of hypoxia in R-18 human melanoma xenografts