Tumor growth requires formation of new blood vessels, a process known as angiogenesis. New blood vessels supply the growing tumor with oxygen and nutrients. Consequently, inhibiting angiogenesis may starve cancer cells and prevent cancer progression.
1Tumor angiogenesis: therapeutic implications.
Inhibiting angiogenesis is an attractive clinical strategy for two reasons. First, all solid tumors, irrespective of histology or mutation profile, are dependent on angiogenesis. Therefore, angiogenesis inhibition could be universally applicable. Second, adverse effects would be expected to be mild because physiological angiogenesis is relatively rare in adults.
2Angiogenesis in life, disease and medicine.
Unfortunately, clinical trials with angiogenesis inhibitors have been disappointing.
3Antiangiogenic therapy: impact on invasion, disease progression, and metastasis.
,4Modes of resistance to anti-angiogenic therapy.
One possible explanation for the limited effect of current angiogenesis inhibitors is that only one type of angiogenesis is targeted when there are, in fact, at least two types of angiogenesis.
5- De Spiegelaere W.
- Casteleyn C.
- Van den Broeck W.
- Plendl J.
- Bahramsoltani M.
- Simoens P.
- Djonov V.
- Cornillie P.
Intussusceptive angiogenesis: a biologically relevant form of angiogenesis.
In contrast to sprouting angiogenesis, intussusceptive angiogenesis is understudied and remains enigmatic. Intussusceptive angiogenesis is a remodeling process in which one vessel splits into two parallel vessels. Intussusceptive angiogenesis starts with the formation of a slender endothelial pillar through the vessel lumen. The pillars widen and merge to form a wall through the vessel that divides the single lumen into two parallel lumens.
9Intussusceptive angiogenesis--the alternative to capillary sprouting.
Intussusceptive angiogenesis was first described in 1986 as a mechanism that rapidly expands the vascular networks in the lungs of postnatal rats.
10- Caduff J.H.
- Fischer L.C.
- Burri P.H.
Scanning electron microscope study of the developing microvasculature in the postnatal rat lung.
,11A novel mechanism of capillary growth in the rat pulmonary microcirculation.
Since then, intussusceptive angiogenesis has been demonstrated in a range of organs during embryonic and postnatal growth,
12A new mechanism of blood vessel growth - hope for new treatment strategies.
in chronic inflammation,
13- Konerding M.A.
- Turhan A.
- Ravnic D.J.
- Lin M.
- Fuchs C.
- Secomb T.W.
- Tsuda A.
- Mentzer S.J.
Inflammation-induced intussusceptive angiogenesis in murine colitis.
,14- Dimova I.
- Hlushchuk R.
- Makanya A.
- Styp-Rekowska B.
- Ceausu A.
- Flueckiger S.
- Lang S.
- Semela D.
- Le Noble F.
- Chatterjee S.
- Djonov V.
Inhibition of Notch signaling induces extensive intussusceptive neo-angiogenesis by recruitment of mononuclear cells.
and in lungs of patients with coronavirus disease 2019–induced respiratory failure.
15- Ackermann M.
- Verleden S.E.
- Kuehnel M.
- Haverich A.
- Welte T.
- Laenger F.
- Vanstapel A.
- Werlein C.
- Stark H.
- Tzankov A.
- Li W.W.
- Li V.W.
- Mentzer S.J.
- Jonigk D.
Pulmonary vascular endothelialitis, thrombosis, and angiogenesis in Covid-19.
The role of intussusceptive angiogenesis in human cancer is almost completely unknown, but it has been described in cancer models.
16- Oliveira de Oliveira L.B.
- Faccin Bampi V.
- Ferreira Gomes C.
- Braga da Silva J.L.
- Encarnação Fiala Rechsteiner S.M.
Morphological characterization of sprouting and intussusceptive angiogenesis by SEM in oral squamous cell carcinoma.
, 17- Ackermann M.
- Morse B.A.
- Delventhal V.
- Carvajal I.M.
- Konerding M.A.
Anti-VEGFR2 and anti-IGF-1R-Adnectins inhibit Ewing's sarcoma A673-xenograft growth and normalize tumor vascular architecture.
, 18- Bugyik E.
- Dezso K.
- Reiniger L.
- László V.
- Tóvári J.
- Tímár J.
- Nagy P.
- Klepetko W.
- Döme B.
- Paku S.
Lack of angiogenesis in experimental brain metastases.
Interestingly, intussusceptive angiogenesis may not rely on VEGF and has even been shown to increase during VEGF inhibition in experimental tumors.
19- Hlushchuk R.
- Riesterer O.
- Baum O.
- Wood J.
- Gruber G.
- Pruschy M.
- Djonov V.
Tumor recovery by angiogenic switch from sprouting to intussusceptive angiogenesis after treatment with PTK787/ZK222584 or ionizing radiation.
,20- Paku S.
- Dezso K.
- Bugyik E.
- Tóvári J.
- Tímár J.
- Nagy P.
- Laszlo V.
- Klepetko W.
- Döme B.
A new mechanism for pillar formation during tumor-induced intussusceptive angiogenesis: inverse sprouting.
Materials and Methods
Ethics
All research was performed in accordance with the Declaration of Helsinki of the World Medical Association. Study approval was obtained from the Gothenburg Regional Ethics Committee, and patients gave written informed consent to participate (numbers 151-16 and 288-12). Animal studies were performed in accordance with European Union directive 2010/63 and approved by the Animal Ethics Committee at the University of Gothenburg (number 36-2014).
Human Melanoma Metastases
Fourteen biopsies of cutaneous melanoma metastases were obtained from patients treated for metastatic melanoma at Sahlgrenska University Hospital (Gothenburg, Sweden). Of these, six biopsies (
Table 1) were paraffin embedded, sectioned with 6-μm thickness, and stained with hematoxylin and eosin and immunofluorescence. An additional eight surgical biopsies were collected for more detailed three-dimensional analyses. These biopsies were embedded in OCT, flash frozen in liquid nitrogen, and stored at −80°C. The frozen biopsies were sectioned into 20- or 40-μm thickness and stained with immunofluorescence.
Table 1Clinical Data of Cutaneous Human Melanoma Metastases Analyzed for Pillars
F, female; M, male; BRAF, v-raf murine sarcoma viral oncogene homolog B1.
Patient-Derived Melanoma Xenografts in Mice and BrafCA/+Ptenf/fTyr-Cre+/0Mice
To establish patient-derived melanoma xenografts (PDXs), melanoma cells from human metastases were mixed with Matrigel (Thermo Fisher Scientific, Waltham, MA) and injected subcutaneously into the flanks of nonobese severe combined immune-deficient IL-2 chain receptor γ knockout mice (Taconic, Ry, Denmark) to form xenografts.
23- Einarsdottir B.O.
- Bagge R.O.
- Bhadury J.
- Jespersen H.
- Mattsson J.
- Nilsson L.M.
- Truvé K.
- López M.D.
- Naredi P.
- Nilsson O.
- Stierner U.
- Ny L.
- Nilsson J.A.
Melanoma patient-derived xenografts accurately model the disease and develop fast enough to guide treatment decisions.
BrafCA/+Ptenf/fTyr
-Cre+/0mice (BPT mice) is a model in which a genetic trigger is activated to generate primary tumors in the skin, after which spontaneous lymph node metastases develop.
24- Dankort D.
- Curley D.P.
- Cartlidge R.A.
- Nelson B.
- Karnezis A.N.
- Damsky Jr., W.E.
- You M.J.
- DePinho R.A.
- McMahon M.
- Bosenberg M.
Braf(V600E) cooperates with Pten loss to induce metastatic melanoma.
,25- Le Gal K.
- Ibrahim M.X.
- Wiel C.
- Sayin V.I.
- Akula M.K.
- Karlsson C.
- Dalin M.G.
- Akyürek L.M.
- Lindahl P.
- Nilsson J.
- Bergo M.O.
Antioxidants can increase melanoma metastasis in mice.
Paraffin sections (6 μm thick) from flank tumors in the PDX model (
n = 6) and lymph node metastases from BPT mice (
n = 8) were obtained and stained with hematoxylin and eosin and processed for immunofluorescence.
Histology and Immunofluorescence
Paraffin-embedded sections were deparaffinized, and heat-induced epitope retrieval in citrate was performed. Frozen sections were fixed in 2% paraformaldehyde for 5 minutes. Sections were either stained with hematoxylin and eosin or labeled using immunofluorescence. Some sections used for immunofluorescence were permeabilized in 0.1% Triton X-100 for 5 minutes, and blocked with 1% bovine serum albumin and 0.3 mol/L glycine in phosphate-buffered saline for 30 minutes. Avidin/streptavidin blocking was performed, and primary antibodies were added (
Table 2) in 4°C overnight. After washing, secondary antibodies (
n = 3) were added for 2 hours, followed by additional washing and mounting of sections with Prolong Gold (Thermo Fisher Scientific). Other sections were stained using a multiplex immunofluorescence assay (Opal Multiplex staining system; Akoya Biosciences, Marlborough, MA), after deparaffinization, antigen retrieval (heat-induced epitope retrieval in Tris buffer), and protein blocking (3% peroxidase and 2% bovine serum albumin). Primary antibodies (
Table 2), diluted in 2% bovine serum albumin, were added to the tissue samples for 60 minutes at room temperature on a movable plate. Following a wash in Tris-buffered saline buffer, the slides were incubated with the secondary horseradish peroxidase conjugate for 10 minutes. After a new washing cycle, the slides were incubated with the corresponding Opal fluorophore diluted in ready-to-use tyramide amplification buffer for 10 minutes. The slides were then put into a plastic holder containing Tris-buffered saline buffer and placed in the microwave at 800 W for 40 seconds, followed by 90 W for 15 minutes. When the slides had cooled to room temperature, they were washed and underwent a new cycle of primary antibody staining. Slides were counterstained with DAPI once all the primary antibody cycles were finished. Following another round of washing, the slides were mounted with Prolong Gold.
Table 2Antibodies Used for Immunofluorescence Staining of Human and Mouse Material
MMP, matrix metalloproteinase; N/A, not applicable.
Stained sections were imaged with a Metafer Slide Scanning Platform (MetaSystems GmbH, Altlussheim, Germany) equipped with custom fluorescence filters.
26- Kijani S.
- Yrlid U.
- Heyden M.
- Levin M.
- Borén J.
- Fogelstrand P.
Filter-dense multicolor microscopy.
Screening for Intravascular Pillars
High-resolution tumor images were visually inspected for intravascular pillars using VsViewer version 2.1.113 (MetaSystems GmbH). Pillars were identified as endothelial structures with a collagen core and/or α-actin–positive cells surrounded by endothelium. In human metastases and PDXs, all vessels located intratumorally and peritumorally (250 μm beyond the tumor margin) were individually visually inspected for the presence of intravascular pillars. To distinguish true pillars from folds or artifacts, cross-sections of suspected pillars were performed using confocal microscopy. Tumor area (mm2) and vessel and pillar densities (number/mm2) were quantified for all samples.
Gene Expression Analysis
RNA was prepared from patient (
n = 11) and PDX biopsies (
n = 26), as described previously.
23- Einarsdottir B.O.
- Bagge R.O.
- Bhadury J.
- Jespersen H.
- Mattsson J.
- Nilsson L.M.
- Truvé K.
- López M.D.
- Naredi P.
- Nilsson O.
- Stierner U.
- Ny L.
- Nilsson J.A.
Melanoma patient-derived xenografts accurately model the disease and develop fast enough to guide treatment decisions.
,27- Einarsdottir B.O.
- Karlsson J.
- Söderberg E.M.V.
- Lindberg M.F.
- Funck-Brentano E.
- Jespersen H.
- Brynjolfsson S.F.
- Olofsson Bagge R.
- Carstam L.
- Scobie M.
- Koolmeister T.
- Wallner O.
- Stierner U.
- Berglund U.W.
- Ny L.
- Nilsson L.M.
- Larsson E.
- Helleday T.
- Nilsson J.A.
A patient-derived xenograft pre-clinical trial reveals treatment responses and a resistance mechanism to karonudib in metastatic melanoma.
,28- Ny L.
- Rizzo L.Y.
- Belgrano V.
- Karlsson J.
- Jespersen H.
- Carstam L.
- Bagge R.O.
- Nilsson L.M.
- Nilsson J.A.
Supporting clinical decision making in advanced melanoma by preclinical testing in personalized immune-humanized xenograft mouse models.
Alignment and Preprocessing of RNA-Sequencing Data
RNA reads were aligned to the hg38 human and to the GRCm38 mouse reference genome assembly using STAR version 2.7.1a,
29- Dobin A.
- Davis C.A.
- Schlesinger F.
- Drenkow J.
- Zaleski C.
- Jha S.
- Batut P.
- Chaisson M.
- Gingeras T.R.
STAR: ultrafast universal RNA-seq aligner.
with splice junctions supplied from the hg38 GENCODE2
30- Harrow J.
- Frankish A.
- Gonzalez J.M.
- Tapanari E.
- Diekhans M.
- Kokocinski F.
- et al.
GENCODE: the reference human genome annotation for the ENCODE Project.
version 27 human and GENCODE version M22 mouse reference annotation, respectively, using the parameters “--twopassMode Basic -outSAMmapqUnique 60” and “--sjdbOverhang 75” or “--sjdbOverhang 125”, depending on sequencing batch. For PDX samples, reads deriving from human were retrieved using Disambiguate version 1.0,
31- Ahdesmäki M.J.
- Gray S.R.
- Johnson J.H.
- Lai Z.
Disambiguate: an open-source application for disambiguating two species in next generation sequencing data from grafted samples.
with the parameter “-a star”.
Estimation of Gene Expression Levels
Aligned reads were binned to genes using htseq-count, (HTSeq version 0.11.2),
32- Anders S.
- Pyl P.T.
- Huber W.
HTSeq--a Python framework to work with high-throughput sequencing data.
with respect to the GENCODE version 27 reference genome annotation, using the parameters “-r name -q -f bam -m intersection-strict” and “-s reverse”, “-s yes,” or “-s no”, depending on sequencing batch.
MMP9, Macrophages, and T Cells in Human and Mice Melanoma
High-resolution tumor images were analyzed to quantify matrix metalloproteinase 9 (MMP9), macrophages, and T cells adjacent to blood vessels with or without pillars in human melanoma metastases. First, blood vessels with verified pillars were identified. The blood vessels were analyzed for proliferating endothelial cells, defined as colocalization of endothelial cell marker ulex europaeus agglutinin I (UEA-1) and Ki-67 staining. MMP9, macrophages, and T cells were quantified in an area around the specified pillar containing vessels, corresponding to a region of interest of 1.23 mm2. Areas without pillars, nonpillar zones, were analyzed in the same way. In mice tumors, MMP9, macrophages, and T cells adjacent to blood vessels were analyzed using the same technique.
In Vitro Models of Pillar and Tube Formation
Two cell co-culture models were used to study either pillar or tube and tip cell formation
in vitro.
33- Levin M.
- Ewald A.J.
- McMahon M.
- Werb Z.
- Mostov K.
A model of intussusceptive angiogenesis.
The first model has features of intussusceptive angiogenesis (pillars) and the second model has features of both vasculogenesis (
de novo formation of blood vessels) and sprouting angiogenesis (tip cell formation). In both models, human pulmonary artery smooth muscle cells (100,000 cells/cm
2; Lonza number CC-2581; Thermo Fisher Scientific) were seeded onto a Transwell polyester membrane insert (number 3460; Corning, Corning, NY). After 24 hours, telomerase-immortalized microvascular endothelial cells (ATCC CRL-4025; ATCC, Manassas, VA) were added on top of the smooth muscle cells. A high density of telomerase-immortalized microvascular endothelial cells (50,000/cm
2) was added on top of the smooth muscle cells to form pillars, and a low density of telomerase-immortalized microvascular endothelial cells (10,000/cm
2) was added to form tubes and tip cells. Medium (EGM2-MV; Lonza, CC-3202; Thermo Fisher Scientific) was changed every second day. The membranes with cells were fixed in 4% paraformaldehyde for 30 minutes, and stored in 0.1% paraformaldehyde until staining of whole filters in the same manner as described above, with the addition of 0.1% saponin during antibody incubations. To investigate the effect of broad-spectrum MMP inhibition on pillar formation, MMP inhibitor batimastat (BB94; British Biotech, Oxford, UK) or ilomastat (Sigma Aldrich, St. Louis, MO) was added to the cell culture medium.
Expression of MMP mRNAs in Pillar Co-Culture Model
mRNA expression of MMPs was analyzed in pillar co-cultures as well as in single cultures of smooth muscle cells or endothelial cells. Isolation of mRNA was performed after 7 days using an RNeasy minikit (Qiagen, Hilden, Germany). cDNA was produced using a High-Capacity cDNA reverse transcription kit (4368813; Applied Biosystems, Foster, CA) with random primers. mRNA expression of the genes of interest was analyzed through a TaqMan real-time PCR in a ViiA 7 system (Thermo Fisher Scientific) with the primers listed in
Table 3, using ActinBeta (ACTB) as an internal control.
Table 3TaqMan Probes Used for qPCR
ACTB, ActinBeta; ID, identifier; MMP, matrix metalloproteinase; qPCR, real-time quantitative PCR.
Live Cell Confocal Microscopy
To investigate if MMP inhibition reduces movement of smooth muscle cells in pillars, live-cell confocal microscopy was used. Smooth muscle cells were marked with Celltracker orange (Thermo Fisher Scientific), and endothelial cells were marked with Celltracker green (Thermo Fisher Scientific). The cells were grown in 96-well cover glass bottom plates (Thermo Fisher Scientific) for 7 days to form pillars. Then, MMP inhibitor batimastat (50 μmol/L; Sigma Aldrich) or vehicle (dimethyl sulfoxide) was added, and smooth muscle cell movement during 24 hours was quantified using live-cell imaging. Live-cell imaging was performed on a custom spinning disk confocal microscope system (Solamere Technology Group, Salt Lake City, UT). Briefly, the system was built around a Zeiss Axiovert 200 M microscope (Zeiss, Oberkochen, Germany), a Yokogawa CSU-10 confocal (Yokogawa, Sugar Land, TX), argon and krypton lasers from Dynamic Lasers (Salt Lake City, UT), a Blue Sky 405 solid state laser (Blue Skye Research, Milpitas, CA), an Applied Scientific Instruments (Eugene, OR) MS-2000 motorized stage, and an Applied Scientific Instruments FW-1000 filter wheel. Zeiss Fluar (10× to 40×), LD-Plan Neofluar (20× to 40×), and LD-LCI C-Apochromat (40×) objective lenses were used (Carl Zeiss, Oberkochen, Germany). Image collection relied on QED In Vivo (Media Cybernetics, Rockville, MD). Image analysis was performed using Imaris version 4.1 (Bitplane, Belfast, UK). Environmental control was achieved using a custom-built enclosure and a World Precision Instruments Air Therm ATX (Sarasota, FL).
Statistical Analysis
The
t-test for unpaired data or analysis of variance with Tukey multiple comparisons or Holm-Šídák test was performed to assess significance, and
P < 0.05 was considered significant. Differentially expressed genes between human biopsies and PDX samples were assessed using DESeq2 version 1.26.0
34- Love M.I.
- Huber W.
- Anders S.
Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.
with the parameter “alpha = 0.05” in R version 3.6.1 (R Foundation for Statistical Computing, Vienna, Austria;
https://www.r-project.org), accounting for sequencing batch. False discovery rate–adjusted
P values were calculated using the Benjamini-Hochberg method, and
P < 0.05 was considered statistically significant.
Discussion
This study presents evidence of intussusceptive angiogenesis in human melanoma metastases. Intraluminal pillars, the hallmark of intussusceptive angiogenesis, were found within blood vessels in human metastases and had similar structure, as shown in embryonic and pathologic angiogenesis.
10- Caduff J.H.
- Fischer L.C.
- Burri P.H.
Scanning electron microscope study of the developing microvasculature in the postnatal rat lung.
,11A novel mechanism of capillary growth in the rat pulmonary microcirculation.
,20- Paku S.
- Dezso K.
- Bugyik E.
- Tóvári J.
- Tímár J.
- Nagy P.
- Laszlo V.
- Klepetko W.
- Döme B.
A new mechanism for pillar formation during tumor-induced intussusceptive angiogenesis: inverse sprouting.
,35- Djonov V.
- Schmid M.
- Tschanz S.A.
- Burri P.H.
Intussusceptive angiogenesis: its role in embryonic vascular network formation.
,36Intussusceptive microvascular growth, a new mechanism of capillary network formation.
Tumor angiogenesis has been an intense area of research for many decades, but there is surprisingly limited knowledge on how new blood vessels are formed within human tumors. Sprouting is often assumed to be the dominating angiogenic mechanism, but new blood vessels could also form via intussusceptive angiogenesis.
37- Nico B.
- Crivellato E.
- Guidolin D.
- Annese T.
- Longo V.
- Finato N.
- Vacca A.
- Ribatti D.
Intussusceptive microvascular growth in human glioma.
, 38- Ribatti D.
- Nico B.
- Floris C.
- Mangieri D.
- Piras F.
- Ennas M.G.
- Vacca A.
- Sirigu P.
Microvascular density, vascular endothelial growth factor immunoreactivity in tumor cells, vessel diameter and intussusceptive microvascular growth in primary melanoma.
, 39- Zhang Z.
- Zhao M.
- Xu Z.
- Song Z.
Angioarchitecture and CD133 + tumor stem cell distribution in intracranial hemangiopericytoma: a comparative study with meningioma.
, 40- Nowak-Sliwinska P.
- Alitalo K.
- Allen E.
- Anisimov A.
- Aplin A.C.
- Auerbach R.
- et al.
Consensus guidelines for the use and interpretation of angiogenesis assays.
, 41- Burri P.H.
- Hlushchuk R.
- Djonov V.
Intussusceptive angiogenesis: its emergence, its characteristics, and its significance.
Intussusceptive angiogenesis may be underestimated because intraluminal pillars are not detected by conventional microscopy. Previously, intravascular pillars in tumors have mainly been visualized in experimental models using advanced three-dimensional reconstructions of serial ultrathin sections or vascular cast techniques.
5- De Spiegelaere W.
- Casteleyn C.
- Van den Broeck W.
- Plendl J.
- Bahramsoltani M.
- Simoens P.
- Djonov V.
- Cornillie P.
Intussusceptive angiogenesis: a biologically relevant form of angiogenesis.
,9Intussusceptive angiogenesis--the alternative to capillary sprouting.
,14- Dimova I.
- Hlushchuk R.
- Makanya A.
- Styp-Rekowska B.
- Ceausu A.
- Flueckiger S.
- Lang S.
- Semela D.
- Le Noble F.
- Chatterjee S.
- Djonov V.
Inhibition of Notch signaling induces extensive intussusceptive neo-angiogenesis by recruitment of mononuclear cells.
,16- Oliveira de Oliveira L.B.
- Faccin Bampi V.
- Ferreira Gomes C.
- Braga da Silva J.L.
- Encarnação Fiala Rechsteiner S.M.
Morphological characterization of sprouting and intussusceptive angiogenesis by SEM in oral squamous cell carcinoma.
,17- Ackermann M.
- Morse B.A.
- Delventhal V.
- Carvajal I.M.
- Konerding M.A.
Anti-VEGFR2 and anti-IGF-1R-Adnectins inhibit Ewing's sarcoma A673-xenograft growth and normalize tumor vascular architecture.
,19- Hlushchuk R.
- Riesterer O.
- Baum O.
- Wood J.
- Gruber G.
- Pruschy M.
- Djonov V.
Tumor recovery by angiogenic switch from sprouting to intussusceptive angiogenesis after treatment with PTK787/ZK222584 or ionizing radiation.
,41- Burri P.H.
- Hlushchuk R.
- Djonov V.
Intussusceptive angiogenesis: its emergence, its characteristics, and its significance.
In the current study, pillars were demonstrated and quantified using a combination of more widely available epifluorescence and confocal microscopy. Using this approach, pillars were detected in high numbers in human melanoma metastases. More important, there are very few previous studies on intussusceptive angiogenesis in human tumors.
37- Nico B.
- Crivellato E.
- Guidolin D.
- Annese T.
- Longo V.
- Finato N.
- Vacca A.
- Ribatti D.
Intussusceptive microvascular growth in human glioma.
, 38- Ribatti D.
- Nico B.
- Floris C.
- Mangieri D.
- Piras F.
- Ennas M.G.
- Vacca A.
- Sirigu P.
Microvascular density, vascular endothelial growth factor immunoreactivity in tumor cells, vessel diameter and intussusceptive microvascular growth in primary melanoma.
, 39- Zhang Z.
- Zhao M.
- Xu Z.
- Song Z.
Angioarchitecture and CD133 + tumor stem cell distribution in intracranial hemangiopericytoma: a comparative study with meningioma.
In one of these previous studies, phase contrast microscopy demonstrated intraluminal tissue folds, a histologic feature indicating intussusceptive angiogenesis, extending across the blood vessel lumen in primary melanomas.
38- Ribatti D.
- Nico B.
- Floris C.
- Mangieri D.
- Piras F.
- Ennas M.G.
- Vacca A.
- Sirigu P.
Microvascular density, vascular endothelial growth factor immunoreactivity in tumor cells, vessel diameter and intussusceptive microvascular growth in primary melanoma.
Interestingly, intraluminal tissue folds were more commonly detected in thick than in thin primary melanomas. Thick melanomas are more aggressive and prone to metastasize, suggesting that intussusceptive angiogenesis may be linked to a more aggressive tumor biology.
Intussusceptive and sprouting angiogenesis are probably complementary, rather than independent, processes in tumor development and progression. Sprouting allows introduction of blood vessels into an avascular space.
20- Paku S.
- Dezso K.
- Bugyik E.
- Tóvári J.
- Tímár J.
- Nagy P.
- Laszlo V.
- Klepetko W.
- Döme B.
A new mechanism for pillar formation during tumor-induced intussusceptive angiogenesis: inverse sprouting.
,42- Makanya A.N.
- Stauffer D.
- Ribatti D
- Burri P.H.
- Djonov V.
Microvascular growth, development, and remodeling in the embryonic avian kidney: the interplay between sprouting and intussusceptive angiogenic mechanisms.
,43- Makanya A.N.
- Hlushchuk R.
- Djonov V.
Intussusceptive angiogenesis and its role in vascular morphogenesis, patterning, and remodeling.
Once the basic vascular network is formed by sprouting, an angioadaptive switch to intussusceptive angiogenesis occurs.
44- Hlushchuk R.
- Makanya A.N.
- Djonov V.
Escape mechanisms after antiangiogenic treatment, or why are the tumors growing again?.
During intussusceptive angiogenesis, single vessels divide into two new vessels by the insertion and expansion of intraluminal pillars. In this way, the initial plexus expands and remodels into a larger vascular network.
35- Djonov V.
- Schmid M.
- Tschanz S.A.
- Burri P.H.
Intussusceptive angiogenesis: its role in embryonic vascular network formation.
,42- Makanya A.N.
- Stauffer D.
- Ribatti D
- Burri P.H.
- Djonov V.
Microvascular growth, development, and remodeling in the embryonic avian kidney: the interplay between sprouting and intussusceptive angiogenic mechanisms.
In agreement with this hypothesis, pillars were predominantly detected in the peripheral zone, including the adjacent stroma, of human melanoma metastases, rather than in the more hypoxic tumor center. Similar peripheral localization of intussusceptive angiogenesis has been reported in a mouse model of colorectal cancer.
20- Paku S.
- Dezso K.
- Bugyik E.
- Tóvári J.
- Tímár J.
- Nagy P.
- Laszlo V.
- Klepetko W.
- Döme B.
A new mechanism for pillar formation during tumor-induced intussusceptive angiogenesis: inverse sprouting.
In addition, there was significantly lower endothelial cell proliferation in pillar zones compared with nonpillar zones. This observation supports that intussusceptive angiogenesis is present in pillar zones because intussusceptive blood vessels, in contrast to sprouting vessels, are mainly nonproliferative.
5- De Spiegelaere W.
- Casteleyn C.
- Van den Broeck W.
- Plendl J.
- Bahramsoltani M.
- Simoens P.
- Djonov V.
- Cornillie P.
Intussusceptive angiogenesis: a biologically relevant form of angiogenesis.
,9Intussusceptive angiogenesis--the alternative to capillary sprouting.
A higher expression of MMP9 mRNA was detected in human pillar-rich metastases than in pillar-poor PDXs. More important, the presence of MMP9 protein adjacent to and inside of pillars was also verified. MMPs are master regulators of remodeling
45- Bonnans C.
- Chou J.
- Werb Z.
Remodelling the extracellular matrix in development and disease.
and have previously been implicated in both intussusceptive and sprouting angiogenesis.
46- Ackermann M.
- Stark H.
- Neubert L.
- Schubert S.
- Borchert P.
- Linz F.
- Wagner W.L.
- Stiller W.
- Wielpütz M.
- Hoefer A.
- Haverich A.
- Mentzer S.J.
- Shah H.R.
- Welte T.
- Kuehnel M.
- Jonigk M.
Morphomolecular motifs of pulmonary neoangiogenesis in interstitial lung diseases.
,47- Esteban S.
- Clemente C.
- Koziol A.
- Gonzalo P.
- Rius C.
- Martínez F.
- Linares P.M.
- Chaparro M.
- Urzainqui A.
- AndrésV
- Seiki M.
- Gisbert J.P.
- Arroyoet A.G.
Endothelial MT1-MMP targeting limits intussusceptive angiogenesis and colitis via TSP1/nitric oxide axis.
This led us to hypothesize that MMPs are important in pillar formation. MMP inhibition decreased pillar formation
in vitro. In contrast, MMP inhibition had no effect on sprouting angiogenesis and vasculogenesis (
de novo formation of blood vessels)
in vitro. Furthermore, live-cell analysis showed that MMP inhibition reduced the migration of smooth muscle cells through established pillars. Accordingly, MMP inhibition may prevent the basement membrane remodeling and smooth muscle migration required for pillar formation. In addition to MMP9, mRNA levels of MMPs 2, 7, and 11 were expressed nominally higher in human tumors, but the difference was not significant. Data from genetically modified mice have established that knockout of a single MMP is often compensated by increased activity of other MMPs.
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Remodelling the extracellular matrix in development and disease.
For example, knockout of MMP9 is compensated by increased MMP2 activity. Both MMP9 and MMP2 degrade basement membrane protein collagen IV and may thus be required for basement membrane remodeling during intussusceptive angiogenesis. As a further level of complexity, there is also cross talk between MMPs (eg, MMP14/membrane type-1 (MT-1), an important activator of MMP2). Because of such compensations and cross talk, broad-spectrum MMP inhibitors targeting many MMPs simultaneously are probably required to inhibit intussusceptive angiogenesis.
MMPs have recently been shown to have an additional role in intussusceptive angiogenesis that is not related to matrix remodeling. In a mouse-colitis model, endothelial cell MMP14/MT1-MMP cleaves thrombospondin-1 to generate C-terminal fragments of thrombospondin-1.
47- Esteban S.
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Endothelial MT1-MMP targeting limits intussusceptive angiogenesis and colitis via TSP1/nitric oxide axis.
These fragments bind αvβ3 integrin and lead to nitric oxide production, which induces vasodilation in arterioles and thus initiates intussusceptive angiogenesis. This observation further supports the rationale to use MMP inhibitors to block intussusceptive angiogenesis. However, it will be essential to establish animal melanoma models to directly investigate if MMP inhibition blocks intussusceptive angiogenesis
in vivo.
Both human and experimental evidence suggests that inflammation promotes intussusceptive angiogenesis.
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The study found higher degrees of intussusceptive angiogenesis in human tumors, with more intratumoral inflammation, compared with mouse tumors with less inflammation. In human metastases, both macrophages and T cells were detected in higher numbers in close proximity to pillars compared with blood vessels without pillars. These results suggest that macrophages and T cells may promote pillar formation. These results are thus in line with previously published observations. However, the exact mechanism explaining how macrophages and T cells promote intussusceptive angiogenesis remains to be determined. One contributing factor may be MMP9 production by macrophages, as macrophages often are a major source of MMP9 in tumors.
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There are several limitations to the study. Pillar quantification was performed on thin tissue sections, and only pillars with a confirmed three-dimensional structure verified with confocal microscopy were included. Pillars torn by sectioning and small endothelial pillars in blood vessels without smooth muscle cells, such as postcapillary venules, were not counted. Therefore, the technique used is likely to underestimate the true number of pillars. However, this would not influence the relative differences in pillars between human metastases and mouse tumors. Finally, the
in vitro system used to study pillar formation has several limitations. The model lacks important components that are likely to initiate pillar formation
in vivo, such as immune cells and blood flow. There is solid evidence indicating an important role of flow hemodynamics in intussusceptive angiogenesis.
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,9Intussusceptive angiogenesis--the alternative to capillary sprouting.
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Inhibition of Notch signaling induces extensive intussusceptive neo-angiogenesis by recruitment of mononuclear cells.
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In particular, flow-induced release of nitric oxide from endothelial cells seems to be essential for initiation of intussusception
in vivo. In our model, pillars form spontaneously and in the absence of flow. Therefore, the
in vitro model used herein is not suitable to define factors that initiate pillar formation.
In vivo models would be needed to define those factors. The strength of our model is that it constitutes a flexible system to study the mechanics of pillar assembly, such as the matrix remodeling and cell movements described in this article.
In conclusion, the findings in this study suggest that intussusceptive angiogenesis may contribute to the growth of human melanoma metastases. Furthermore, MMP inhibition may be a new therapeutic strategy to inhibit intussusceptive angiogenesis. Combined targeting of intussusceptive angiogenesis and sprouting angiogenesis should be further explored as a treatment option in therapy-resistant metastatic melanomas.
Article info
Publication history
Published online: August 13, 2021
Accepted:
July 26,
2021
Footnotes
Supported by the Swedish state under the agreement between the Swedish government and the county councils, the Avtal om Läkarutbildning och Forskning (ALF) agreement (M.L., M.O.B., J.A.N., and S.B.), Jubileumsklinikens Cancerfond, Lions Cancerfond, Stiftelsen Tornspiran, Kungliga och Hvitfeldtska Stiftelsen, Stiftelsen Assar Gabrielssons Fond, Wilhelm och Martina Lundgrens Vetenskapsfond, Serena Ehrenströms Fond, Torsten och Sara Janssons Fond, and NIH National Institute of Diabetes and Digestive and Kidney Diseases grant R01DK74398 (K.E.M.).
Disclosures: None declared.
Copyright
© 2021 American Society for Investigative Pathology. Published by Elsevier Inc.